Effects Of Intragastric Injection Of Botulinum Toxin A On Gastric Motility, Interstitial Cells Of Cajal,and Expression Of Braingut Peptides(Ghrelin,NPY,and PYY)in Rats

[Background and aims]Obesity is a worldwide health problem. Dietary, pharmacological and behavioral treatments are effective in some of the patients, but the efficacy is temporal if the intervention is interrupted. Surgical treatments (gastric banding and bypass) are effective in some patients, but they are invasive and may induce severe complications. It is important to seek a kind of method with potent efficacy and minimal invasion for obese patients. Recently, as a novel method, intragastric injection of Botulinum Toxin A (BTX-A) is proposed and tried with enthusiasm. Till now, a few reports have showed that intramuscular injections of BTX-A into the gastric wall could induce a reduction in food intake and body weight. These effects might be mediated by delaying gastric emptying or reducing appetite. In China, LIU Qing-sen et al. initially employed BTX-A on the treatment of obesity and confirmed its efficacy in the pilot studies. However the mechanisms are still unclear. In order to explore the possible molecular mechanisms of BTX-A for the treatment of obesity, we investigated the effects of BTX-A injection into different gastric regions for different treatment period, and observed the effects on gastric motility, interstitial cells of Cajal (ICC), and expression of some of the braingut peptides (ghrelin, NPY, PYY) in this study.[Methods]Seventy-two male Wistar rats were randomly assigned to three batches (2-week batch,6-week batch,12-week batch) (24 rats/batch). Then the rats in each batch were assigned to four groups (n=6 rats/group). Control group:saline injection in the gastric wall; antrum group:BTX-A injection in the antrum; fundus group:BTX-A injection in the fundus; multi-site group:BTX-A injection at multiple sites. EACh rat was laparotomized and the stomach was exposed. The BTX-A or saline was injected into the gastric wall individually. After operation, every rat was fed in a separated cage with water and food ad libitum. The food intake and body weight were measured and followed up for 12 weeks. For each batch, gastric motility of rats were tested then samples were harvested at week 2, week 6 or week 12, respectively. The parameters included two parts:part one consisted of gastric motility-related parameters including scintigraphic test of gastric emptying, gastroelectromyogram, expression of ICCs in stomach measured by immunohistochemical analysis, the ultrastructure of ICCs observed by transmission electron microscopy (TEM). Part two consisted of braingut peptides-related parameters such as plasma concentrations of ghrelin and PYY determined by an ELISA analysis, expression of ghrelin and NPY measured by immunohistochemical analysis, Western blotting, and mRNA fluorescent quantitation PCR.[Results]1. The BTX-A treated groups showed a significant reduction of food intake and body weight as compared to the control group.2. The effects of BTX-A on gastric emptying were characterized by a significant increase in half emptying time. Gastroelectromyograms showed bradygastria and rhythm disturbance after expoxure to BTX-A. Expression of ICC and destruction of the ICC net-work structure were noticed in the BTX-A treated groups in comparison to the control group.3. Significant reduction of plasma ghrelin and elevation of plasma PYY was noted in the BTX-A treated groups. Levels of protein and mRNA expression of grelin and NPY were decreased significantly in the BTX-A treated groups as compared to the control group, measured by immunohistochemistry, western blotting and RT-qPCR analysis.4. Significant delaying of gastric emptying, disturbance of gastric electrical rhythm and reduced expression of ICC, ghrelin, and NPY were observed in all the three BTX-A treated groups (Antrum, Fundus, and Multi-site). Among them, the greatest effect was noted in the Multi-site group, followed by Fundus group and Antrum group (P<0.05). The greatest effects were observed in the 2-week batch, and the effects attenuated in the 6-week and 12-week batches (P<0.05)[Conclusions]1. Intragastric injection of BTX-A is proposed to cause the dysmaturity of ICCs and destruction of ICC net-work, to affect the formation and propagation of normal slow-wave, and to disorder the gastric electro-rhythm. This may be one of the important mechanisms of decreasing food intake and body weight in rats.2. In the BTX-A treated groups, down-regulation of appetide-promoting factors (e.g., ghrelin and NPY) and up-regulation of appetide-inhibiting factor (e.g., PYY) were proved. This mechanism may also play a role in reduction of food intake and body weight.3. Among the 4 groups of different injection sites, food intake and body weight reduction were most significantly in the Multi-site group, and the effects were greatest at week 2 among the 3 batches. It was confirmed that the treatment of obesity using BTX-A was effective as long as 12 weeks.In this study, the possible molecular mechanisms of BTX-A in the treatment of obesity was investigated. This may provides more extensive and reliable evidences on the utilization of BTX-A for the treatment of obesity in the future.