Background. Gastric electrical pacing (GEP) could restore interstitial cells of Cajal in diabetic rats. M2 macrophages contribute to the repair of interstitial cells of Cajal injury though secreting heme oxygenase-1 (HO-1). The aim of the study is to investigate the effects and mechanisms of gastric electrical pacing on M2 macrophages in diabetic models. Methods. Sixty male Sprague-Dawley rats were randomized into control, diabetic (DM), diabetic with the sham GEP (DM+SGEP), diabetic with GEP1 (5.5 cpm, 100 ms, 4 mA) (DM+GEP1), diabetic with GEP2 (5.5 cpm, 300 ms, 4 mA) (DM+GEP2), and diabetic with GEP3 (5.5 cpm, 550 ms, 4 mA) (DM+GEP3) groups. The apoptosis of interstitial cells of Cajal and the expression of macrophages were detected by immunofluorescence technique. The expression levels of the Nrf2/HO-1 and NF-κB pathway were evaluated using western blot analysis or immunohistochemical method. Malonaldehyde, superoxide dismutase, and reactive oxygen species were tested to reflect the level of oxidative stress. Results. Apoptosis of interstitial cells of Cajal was increased in the DM group but significantly decreased in the DM+GEP groups. The total number of macrophages was almost the same in each group. In the DM group, M1 macrophages were increased and M2 macrophages were decreased. However, M2 macrophages were dramatically increased and M1 macrophages were reduced in the DM+GEP groups. Gastric electrical pacing improved the Nrf2/HO-1 pathway and downregulated the phosphorylation of NF-κB. In the DM group, the levels of malonaldehyde and reactive oxygen species were elevated and superoxide dismutase was lowered, while gastric electrical pacing reduced the levels of malonaldehyde and reactive oxygen species and improved superoxide dismutase. Conclusion. Gastric electrical pacing reduces apoptosis of interstitial cells of Cajal though promoting M2 macrophages polarization to play an antioxidative stress effect in diabetic rats, which associates with the activated Nrf2/HO-1 pathway and the phosphorylation of NF-κB pathway.
For centuries, gastrointestinal motility disorders, one of the most frequently occurring diseases, have kept perplexed people’s lives with decades of diabetes mellitus . Gastroparesis, namely delayed gastric emptying, is a disorder that slows or reduces the food transit from the stomach to the small intestine without mechanical obstruction. However, effective therapies for gastroparesis remain elusive with limited roles and side effects. Fortunately, nondrug treatments, such as electroacupuncture (EA) and gastric electrical stimulation (GES), are gradually taken seriously because of its apparent efficacy without side effects. In particular, long pulse GES, for it induces slow waves also referred to as gastric electrical pacing (GEP), has a direct impact on gastric motility. At present, GEP develops as an alternative therapy of gastroparesis, but the mechanisms underlying its efficacy remain unclear.
Interstitial cells of Cajal (ICCs) serve as pacemakers and generate slow waves spontaneously in the stomach. Defect of ICCs has been consistently found in both humans and animal models with diabetic gastroparesis [2, 3]. We have reported that long-pulse GES could repair the injured ICCs partly by IGF-1 signaling pathway and enhancing the proliferation of ICCs [4, 5]. However, apoptosis of ICCs was also certificated in the stomach of gastroparesis ; the effects and mechanisms of GEP on apoptosis of ICCs need to be further clarified.
Macrophages have two different phenotypes: proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. They can be transformed into each other in a certain internal environment. Studies have shown that gastrointestinal motility disorder did not occur in diabetic mice with the absence of macrophages, suggesting that macrophages may participate in the development of gastroparesis . It is also reported that phenotypic polarization of M2 macrophages could improve the delayed gastric emptying . Further studies have shown that there was no significant change in the total number of macrophages in animal models and patients of diabetic gastroparesis, while CD206+ M2 macrophages were selectively decreased, accompanied by ICC deletion, resulting in delayed gastric emptying [9–11]. Accordingly, GEP may promote the phenotypic polarization of M2 macrophages to improve ICC expression and gastric emptying.
Heme oxygenase-1 (HO-1) is a widely existing antioxidant defense enzyme, which is not expressed or low expressed in normal tissues, but upregulated during stress playing an anti-inflammatory, antioxidative, and antiapoptotic role. In the gastrointestinal tract, HO-1 is mainly produced and expressed by resident M2 macrophages . Recently, in a study of diabetic gastroparesis model, it has been found that the downregulation of HO-1 could not resist oxidative stress injury, which leads to the destruction of ICCs network and delayed gastric emptying, but upregulation of HO-1 could repair the injury of ICCs and improve the delayed gastric emptying [13, 14]. Similarly, Tian LG et al. reported that the expression of HO-1 in gastric antrum of diabetic gastroparesis mice was significantly decreased, and EA could improve the expression of HO-1 and gastric motility . Therefore, we speculate that GEP may play a key role in improving gastric motility disorder by upregulating HO-1 to repair ICC injury.
Also, studies have shown that HO-1 can protect and reverse oxidative stress damage to ICCs . Stem cell factor (also known as SCF, c-kit ligand) is essential for survival and maintenance of ICCs, but there are few reports about the correlation between HO-1 and SCF. Recent studies  have shown that the NF-κB signaling pathway was activated in diabetic gastrointestinal motility disorders and the decrease expression of SCF/c-kit causes the increase of ICC apoptosis, suggesting that the activation of NF-κB signaling may be an important factor in ICC apoptosis. It is also reported that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in defending against inflammation in different tissues via activation of phase II enzyme HO-1 and inhibition of the NF-κB signaling pathway . Other studies  have shown that HO-1 can inhibit the phosphorylation of NF-κB p65, promote the binding of anti-apoptotic genes to NF-κB, and promote gene transcription to play an antiapoptotic role. In addition, studies in human lung fibroblasts have shown that nuclear transcription factor NF-κB binds to enhancers of SCF gene and promotes SCF transcription . We assumed that HO-1 may play an antiapoptotic role by inhibiting the phosphorylation of NF-κB p65.
In this study, we aimed to explore the effects of GEP on ICC apoptosis and phenotypic polarization of macrophages and to investigate the possible mechanisms of renovation of ICC injury in diabetic rats.
2. Materials and Methods
Male Sprague-Dawley rats (weighing 160-200 g, ) were purchased from Jinan Pengyue Experimental Animal Breeding Co. Ltd. (Shandong, China) and were kept in the suitable laboratory conditions (22-23°C, 12/12 h light-dark cycle) with food and water ad libitum. The experimental procedures were implemented, following the ethical guidelines from the Animal Care and Use Committee of Binzhou Medical University Hospital Laboratory Animal Ethical Committee. The rats were randomly divided into the normal control, diabetes (DM), diabetic+sham GEP (DM+SGEP), and diabetic+GEP (DM+GEP) groups (Figure 1).