Numerous recent studies have demonstrated the ability of curcumin to halt or prevent certain types of cancer, decrease inflammation, and improve cardiovascular health. However, very few studies have examined its ability to protect against diabetic gastroparesis. In our study, we demonstrated that supplementation with curcumin attenuated oxidative stress and NF-κB activation, and prevented the down-regulation of SCF/c-kit protein level in diabetic rats. Our result suggests that the increased gastric dysfunction in diabetic rats can be prevented by curcumin treatment.
Data presented in this study show that SCF/c-kit is down-regulated after the development of diabetes and the concurrent increase in the level of oxidative stress. This down-regulation of SCF/c-kit was lost in all rats that went on to develop delayed gastric emptying and maintained in all rats that did not develop delayed gastric emptying. Failure to maintain down-regulation of SCF/c-kit and develop delayed gastric emptying was associated in all rats with high levels of markers of oxidative stress and NF-κB. In the curcumin-treated rats, ICCs were protected, suggesting that the maintenance of normal gastric function requires decreased ROS, restrained NF-κB activation and improved SCF/Kit expression (e.g. restored to normal levels).
Diabetes is a pathologic condition, and the morbidity and mortality associated with diabetes are the result of myriad complications related to the disease. Oxidative stress plays an important role in the etiology of the disease, and is considered to be the main factor leading to the development of diabetic complications and tissue injury. Diabetes is associated with increased oxidative stress. Oxidative stress mediates many of the deleterious effects of diabetes on organ function, and induces not only gastric mucosal injury, but also gastric motility dysfunction, such as diabetic gastroparesis. Gastroparesis is thought to be caused by ROS-induced damage to the networks formed by ICCs. Moreover, antioxidants can reverse diabetic gastroparesis in NOD mice. ICCs in normal tissues are resistant to oxidative stress caused, for example, by hyperglycemia, but they can become vulnerable to glycemic stress when antioxidant defenses are compromised. The results of our study show the potential mechanisms by which oxidative stress induces gastric motility dysfunction in diabetic rats. Diabetic rats exhibited slower gastric emptying together with increased MDA levels and decreased SOD levels compared with normal rats, with a tight correlation between high levels of MDA and the presence of delayed gastric emptying. Why does oxidative stress damage stomach function? ROS might inflict direct damage to vital cell constituents such as lipids, proteins and DNA, but also modulate patterns of gene expression through functional alterations of transcription factors such as NF-κB. First, mature ICCs are easily damaged under conditions of oxidative stress. ROS are a crucial regulator of cellular signal transduction and energy transmission, and a disturbance of the balance between ROS-generating and ROS-scavenging capabilities might lead to cell damage. Oxidative stress is now recognized as a stimulator of cell responses such as apoptosis. A growing number of studies have described that ROS can activate inflammasomes in cells, leading to increased production of TNF-α, IL-1, IL-6 and IL-18. All sorts of inflammatory factors may directly damage ICCs. We examined the effect of curcumin on ROS formation in stomach tissues. Our finding that ROS content was significantly greater in Group II animals than in Group III animals suggests that curcumin attenuates diabetes-induced ROS formation. Our results also revealed that curcumin is a potent inhibitor of ROS-induced apoptosis. NF-κB is known to be a key factor in up-regulating inflammatory cytokines. NF-κB activation enhances the transcription of pro-inflammatory cytokines, and these cytokines in turn activate NF-κB. A perturbation of NF-κB distribution promoted inflammatory mediator-mediated ICC apoptosis. NF-κB is normally located in cytoplasm where it binds I-κB to form an inactive complex. The phosphorylation and subsequent degradation of I-κB result in NF-κB activation. We found that the NF-κB level in the cytoplasm was higher in diabetic rats than in control rats. Activated NF-κB migrates into the nucleus, and causes the expression of inflammatory cytokines. A comparison of the nuclear NF-κB levels in Group II and III rats revealed that nuclear localization of NF-κB is markedly inhibited by curcumin. We also found that I-κB is degraded in the stomach smooth muscle of diabetic rats. Activation of NF-κB was induced by phosphorylation of the inhibitor IkB, in response to diverse stimuli including ROS, which leads to its degradation and results in unmasking of nuclear localization signals that allow NF-κB to be translocated into the cell nucleus. ROS can directly activate NF-κB by degrading or modifying I-κB in the cytoplasmic NF-κB–IκB complex. The results of this study show that, in rats with experimentally induced gastroparesis, proteolysis of IκB results in activation and nuclear translocation of NF-κB, and that this was accompanied by ROS up-regulation. Previously, various authors have reported that curcumin is a potent inhibitor of transcription factors[34, 35], and that the suppression of NF-κB activation by curcumin results, in turn, in a down-regulation of ROS and inflammation.
It was suggested that loss of ICCs might have a major role in the pathogenesis of diabetic gastroparesis, and that the degree of ICC loss was in proportion to the severity of symptoms or changes in gastric emptying. Curcumin neutralized diabetes-induced oxidative stress, reduced MDA levels, improved SOD levels, restored NF-κB activity, and inhibited inflammatory mediator production, thereby minimizing diabetes-induced ICC apoptosis. All of these observations point toward a crosstalk between a ROS–NF-κB–inflammation mediator in deciding the fate of ICC cells in a high glucose micro-environment, and the intervening role of curcumin.
Reduced SCF levels in the stomachs of diabetic mice have been reported. Because SCF/c-Kit signaling is important for the maintenance of ICC phenotypes, proliferation, and differentiation, we investigated whether ICCs could regenerate after impairment in diabetic rats following curcumin treatment. Furthermore, the kit ligand SCF was mainly observed in the smooth muscle cells (SMCs), which are located close to ICCs. In the proximal stomach, intramuscular ICC rather than myenteric ICC are the main subtypes of ICCs that give rise to gastric slow waves and act as the dominant pacemaker cells. Previous studies have shown that SCF is important for maintaining a steady level of ICCs in stomach tissues. SCF is produced by SMCs in the long-term maintenance of ICCs, while ICC depletion in diabetes is accompanied by smooth-muscle atrophy and reduced SCF levels. Exogenous SCF can partially reverse the pathological changes in ICCs in diabetic mice, because SCF is necessary for differentiation of precursors into ICCs. In our study, we found the SCF/c-kit levels and ICC numbers were clearly declined in diabetic gastroparesis rats compared with control rats. Indeed, the loss of SCF might be responsible for the loss of ICCs. The levels of mRNA expression for SCF and c-kit were lower in the stomach tissues of diabetic rats than non-diabetic rats, and decrease was significantly reversed by the curcumin intervention. These findings showed that curcumin improves SCF/c-kit levels in the stomach tissues of diabetic rats. These results also indicate that the fate of ICCs in diabetes depends on SCF/c-kit from SMCs, but probably not that from myenteric neurons. No differences in fasting blood glucose levels and body weight were noted before and after treatment in the same groups of rats, indicating that the pathologic ICCs changes can be attributed to a deficiency of endogenous SCF/c-kit, and not related to hyperglycemia. We showed that ICC loss owing to diabetes involves reduced expression of SCF/c-kit and a critical differentiation and survival factor for ICCs. Developmental studies have demonstrated that c-kit+ mesenchymal precursor cells appear to generate ICCs and the longitudinal muscle layer. Cell fate decisions between becoming an ICC or a SMC depend on SCF/c-kit signaling. We found that a loss or lack of SCF and c-kit ligand could lead to the apoptosis of ICCs, but when Group II rats were treated with curcumin, we observed an increase in SCF/c-kit concentration in the micro-environment promoting ICC transdifferentiation back into a normal phenotype. Our results suggest that curcumin could aid in the restoration of the SCF/c-kit signaling pathway, which is essential for ICC phenotype restoration and functional recovery. These results reinforce the results of previous studies showing that ICCs displaying distinctive plasticity can be controlled by changing the level of SCF/c-kit signaling.
Overall, our results show that curcumin has an effect on anti-oxidation and free radical removal. We further show that curcumin reduces activation of NF-κB via inhibition of oxidative stress. In addition, our results suggest that curcumin promotes the expression of SCF/c-kit. As a result, we have a better understanding of the molecular mechanism by which curcumin protects ICCs, namely, via blocking of oxidative stress, inhibition of NF-κB activation and an enhancement of SCF/c-kit expression.