Curcumin protects remote organs against injury that is caused by intestinal ischemia and reperfusion

Esta obra está bajo una Licencia Creative Commons Atribución 4.0 Internacional.

Ischemia and reperfusion (I/R) are pathophysiological sequelae that can be caused by various clinical events (Borges et al., 2018). Several diseases, transplantation surgeries, and accidents can result in intestinal I/R. The incidence of death is high for these patients, particularly for those in critical condition, in which the risk of mortality is 60-80% of cases (Hartmann et al., 2017; Ozban et al., 2015).

Injury that is caused by intestinal I/R is associated with numerous local pathophysiological consequences, such as an increase in neutrophil migration, an increase in cytokine release, and a decrease in the myenteric neuronal population in the ileum in rats (Borges, Souza, Beraldi, Schneider, & Buttow, 2016; Lin et al., 2014). One of the most relevant consequences is the lesion of intestinal cells that negatively affects mucosal integrity, leading to an increase in intestinal epithelial permeability (Borges et al., 2018; Eltzschig & Eckle, 2011; Lin et al., 2014). Several studies have suggested that injury to the intestinal mucosa allows the translocation of bacteria and endotoxins from the intestinal lumen into the bloodstream (Borges et al., 2018; Lin et al., 2014; Tassopoulos, Chalkias, Papalois, Iacovidou, & Xanthos, 2017), which can cause the development of lesions in remote organs (Stringa et al., 2016). However, the consequences of intestinal I/R in remote organs are not yet fully understood.

The consequences of intestinal I/R injury that are associated with bacterial translocation (Hebra et al., 1994; Koch, 2016) are generally known to lead to oxidative stress and systemic inflammation (Fan et al., 2014; Koch, 2016; Wang et al., 2015). Initially, during the period of ischemia, several local inflammatory mediators are generated (Eltzschig & Carmeliet, 2011). The reperfusion period, characterized by the return of blood circulation to ischemic tissue, triggers the formation of free radicals in the intestine (Mallick, Yang, Winslet, & Seifalian, 2004). The translocation of bacteria and their byproducts, in addition to promoting inflammation in diverse tissues and organs, leads to an increase in reactive species of oxygen (ROS) and reactive nitrogen species (RNS) (Koch, 2016) and consequently oxidative stress in affected tissues and organs.

Therapeutic measures may prevent or alleviate the pathophysiological changes that are promoted by intestinal I/R injury in the intestine (Borges et al., 2016; Tian et al., 2016) and other organs (Fan et al., 2014; Onder et al., 2012). The use of antioxidants has often been adopted as a treatment alternative with good results (Borges et al., 2016; Souza et al., 2015; Onder et al., 2012; Tassopoulos et al., 2017). Several authors reported that curcumin (diferuloylmethane, 1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione]) has antioxidant and antiinflammatory activity and high therapeutic potential (Gupta, Patchva, Koh, & Aggarwal, 2012; He, Yue, Zheng, Zhang, Chen, & Du, 2015. This substance is an orange-yellow component of saffron (Curcuma longa) (Okudan, Belviranlı, Gökbel, Oz, & Kumak, 2013). Some intestinal I/R injury studies have reported its beneficial effects on the gut (Karatepe et al., 2009; Atalik et al., 2012; Yucel, Kanter, Pergel, Erboga, & Guzel, 2011). However, studies of the effects of curcumin on remote organs that are affected by intestinal I/R are still limited. Therefore, the present study evaluated the effects of curcumin on oxidative and inflammatory parameters in the liver and kidneys in rats that were subjected to 45 min. of intestinal ischemia followed by 7 days of reperfusion.

Aspartate aminotransferase and ALT levels

No significant differences in AST or ALT levels were found between groups (p > 0.05; Table 2).

Table 2
Levels of Aspartate Aminotransferase AST and Alanine Aminotransferase ALT

Results were expressed as mean ± standard error. (n = 6). C = control, SC = sham control, ST = sham treated, IRC = ischemic control, and IRT = ischemic treated. There was no statistically significant difference.

Several therapeutic substances have been tested and applied for the treatment of I/R-related injury (Borges et al., 2016; Souza et al., 2015; Hartmann et al., 2017; Inan et al., 2013; Ozban et al., 2015; Stringa et al., 2016). Curcumin has been extensively studied in recent years. In addition to antioxidant and antiinflammatory activity, curcumin has been reported to exert antidiabetic, antiangiogenic, antimutagenic, antiinfective, and anticancer effects (Aggarwal, Gupta, & Sung, 2013; He, Yue, Zheng, Zhang, Chen, & Du, 2015; Rahmani, Alsahli, Aly, Khan, & Aldebasi, 2018; Kumar, Houreld, & Abrahamse, 2018). Several studies have shown that curcumin has a protective effect on multiple organs, including the kidneys (Fan et al., 2017; Onder et al., 2012; Tian et al., 2016) and liver (Rahmani et al., 2018). We hypothesized that curcumin may reduce or prevent the increase in neutrophil migration (measured as MPO activity) and prevent oxidative stress in the kidneys and liver in rats that are subjected to I/R.

Intestinal ischemia followed by reperfusion may trigger both local pathophysiological changes and cause damage in remote organs. However, little is known about the ways in which remote organs are affected after intestinal I/R injury or the effects of curcumin thereon. In the present study, we observed an increase in MPO activity in the liver in rats that were subjected to intestinal I/R and treated with vehicle, reflecting an increase in neutrophil migration and oxidative damage. Some authors reported that intestinal I/R injury, in addition to promoting oxidative stress in the ileum in rats (Borges et al., 2018; Souza et al., 2015), also compromises the integrity of the epithelial barrier (Ajamieh & Teoh, 2009; Berg, 1999) and may lead to translocation of bacteria and their byproducts, such as lipopolysaccharide and others endotoxins, from the intestinal lumen to the systemic circulation or portal vein (He, Li, Wang, Zhang, Ke, Yan, & Chen, 2015; Hebra et al., 1994; Koch, 2016). Thus, during intestinal I/R injury, the portal vein, in addition to transporting nutrients, also transports microbial products and bacteria (Wang et al., 2015). The liver receives a greater load of molecules during I/R (Wang et al., 2015), thus resulting in greater damage compared with more distant organs. In the present study, the beneficial effects of curcumin were more pronounced in hepatic tissues.

The enzymatic activity of MPO in the liver increased by 72% in liver samples in the IRC group relative to the C group. Similar results were also reported by Fan et al. (2014), in which rats were subjected to intestinal ischemia for 1h followed by 2h of reperfusion. The increase in MPO activity is mainly attributable to the infiltration of neutrophils in the tissue, causing liver inflammation (Fan et al., 2014). In addition to bacterial translocation, bacterial products also contribute to the progression of liver injury through the activation of specific pathogen recognition receptors, especially Toll-like receptor 4 (TLR4) (Schnabl & Brenner, 2014; Wang et al., 2015). Even low levels of activated TLR4 are able to activate Kupffer cells. To eliminate liver pathogens, Kupffer cells trigger an inflammatory cascade that results in the release of cytokines (e.g., interleukin-6 [IL-6], IL-1β, and tumor necrosis factor-α [TNF-a]) (Koch, 2016; Lin et al., 2014) and inflammation (Chassaing, Mesmin, & Gewirtz, 2014; Haque & Barritt, 2016; Koch, 2016). In the present study, oral treatment with curcumin (60 mg kg^-1) attenuated inflammation in the liver after intestinal I/R injury. The IRT group exhibited improvements in MPO activity. Previous studies substantiated the antiinflammatory potential of curcumin, which has the ability to suppress proinflammatory pathways by blocking both the production and action of inflammatory cytokines, such as TNF-α, IL-1, IL-2, IL-6, IL-8, and IL-12 (Aggarwal et al., 2013; Rahmani et al., 2018; Wojcik, Krawczyk, Wojcik, Cypryk, & Wozniak, 2018).

Inflammation and oxidative stress are closely related phenomena (Wojcik et al., 2018). Oxidative stress is related to disturbances in the balance between oxidants and antioxidants, leading to the aberrant control of redox signaling and molecular damage (Sies & Cadenas, 1985). Superoxide dismutase is an enzyme that inactivates reactive species and protects against oxidative stress. The present results showed that SOD activity in the liver increased by 41% in the IRC group compared with the C group. Curcumin has a chemical structure that is capable of eliminating reactive oxygen and nitrogen species (Simioni et al., 2018), acting as a powerful antioxidant (He, Yue, Zheng, Zhang, Chen, & Du, 2015). Treatment with curcumin in the IRT group promoted the restoration of SOD activity to levels that were similar to the C group, thus demonstrating its therapeutic potential.

Lipid peroxidation is a consequence of the accumulation of reactive species (He, Yue, Zheng, Zhang, Chen, & Du, 2015; Kong, Blennerhassett, Heel, McCauley, & Hall, 1998) that favor the promotion of cellular injury and necrosis (Kong et al., 1998). Hartmann et al. (2017) subjected rats to 30 min. of ischemia followed by 15 min. of reperfusion, and Inan et al. (2013) subjected rats to 2h of ischemia followed by 2h of reperfusion. These two studies reported the occurrence of lipid peroxidation in the liver in rats. Our results indicated that LOOH levels remained high, even after 7 days of reperfusion. The IRC group exhibited a 16% increase in LOOH levels compared with the C group. Rahmani et al. (2018) reported that curcumin prevented the formation of lipid peroxides. In the present study, curcumin treatment in the IRT group resulted in LOOH levels that were similar to the C group.

Reduced glutathione is a critical indicator of antioxidant capacity (Franco & Cidlowski, 2012; Ma et al., 2014), playing a key role in defense mechanisms against oxidative stress, acting as a cofactor of glutathione peroxidase (GPx), and participating in the elimination of lipid hydroperoxides (Karabulut et al., 2006). Ma et al. (2014) subjected rats to 45 min. of intestinal ischemia followed by 90 min. of reperfusion and found low GSH levels in the liver. Similarly, in the present study, GSH levels decreased by 19% in the IRC group compared with the C group, but this change was not statistically significant. The SC group exhibited a significant 36% decrease in GSH levels compared with the C group. Curcumin is able to modulate GSH levels (Simioni et al., 2018), which may explain why GSH levels were similar between the ST and IRT groups and the C group.

Glutathione .-transferase is an enzyme that has many functions in the body. In addition to having antioxidant activity and being responsible for the detoxification of xenobiotic compounds (Khurana et al., 2002), GST activity has been related to injury that is caused by I/R (Platz et al., 1997; Schön et al., 1997). We found that GST activity in the liver significantly increased by 38% in the IRC group compared with the C group. Curcumin improves the function of detoxifying enzymes, such as GST (Rahmani et al., 2018). In the present study, the IRT group exhibited GST activity that was similar to the C group.

In the present study, the kidneys presented few alterations of the measured parameters. Significant alterations may have been more evident during an acute period of reperfusion. Kiliç et al. (2012) reported various changes in the rabbit kidneys, such an increase in LOOH levels and MPO activity and a decrease in SOD activity, during an acute reperfusion time of 3h. In the present study, the effects of intestinal I/R were evaluated after 7 days of reperfusion. We found that MPO and SOD activity and GSH and LOOH levels were not significantly different between groups. However, the weight of the kidneys was reduced by 12% and 15% in the SC and IRC groups, respectively. This led us to infer that the lower kidney weight in these groups resulted from injury that was caused by intestinal I/R during the acute period of injury and that the kidneys recovered from these deleterious alterations of biochemical parameters after 7 days of reperfusion. Importantly, however, curcumin treatment also had beneficial effects on the kidneys, in which kidney weight was similar between the ST and IRT groups and C group. Previous studies indicated that after 24h of reperfusion following intestinal ischemia injury, some parameters, such as histopathological changes in the intestine and liver, were restored (Lindeström & Ekblad, 2004; Marosti et al., 2015; Stringa et al., 2012). Similarly, in the present study, AST and ALT levels were not significantly different between groups. One possibility is that only oxidative and inflammatory damage persisted after 7 days of reperfusion.

As a consequence of oxidative and inflammatory damage that intestinal I/R can trigger, systemic inflammatory response syndrome and multiple organ failure can occur (Pacher, Nivorozhkin, & Szabó, 2006; Stallion et al., 2005). Tian et al. (2016) reported that curcumin pretreatment effectively promoted the maintenance of intestinal barrier integrity and prevented pathological damage that were associated with intestinal I/R injury. Curcumin treatment at a dose of 60 mg kg^-1 started 5 days before surgery and was maintained daily for up to 7 days later. This treatment regimen exerted antiinflammatory, antioxidant, and hepatoprotective effects and possibly renoprotective effects in preventing damage that was caused by intestinal I/R.

We found that intestinal I/R mainly affected the liver, promoting inflammation and oxidative stress. At a later time of reperfusion (7 days), oxidative and inflammatory damage persisted, and the kidneys appeared to undergo repair much sooner than the liver. Treatment with curcumin had beneficial effects, ameliorating or even preventing injury that was caused by intestinal I/R in the liver and kidneys in rats.

We thank the Post-Graduation Program in Biological Sciences: Cellular and Molecular Biology of Universidade Estadual de Maringá and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We thank the technicians Maria Eurides Cancino, Maria dos Anjos Fortunato, and Maria Ângela Moreira in the histology laboratory at the Universidade Estadual de Maringá.