Glucagon-Like Peptide-2 Modulates The Effects Of Autophagy On Intestinal Adaption In Short Bowel Syndrome

To date, it is well known that the mesenteric vascular lesions can lead to ischemia and even necrosis of the small intestine, resulting in short bowel syndrome(SBS) through one or multiple surgical resection of the affected bowel. Ischemia-reperfusion injury(IRI) is defined as a process in which injury is worse during reperfusion following intestinal ischemia. It occurs commonly in SBS caused by the mesenteric vascular lesions.Autophagy is a controlled and orchestrated process in all prokaryotic and eukaryotic cells,whereby cells “eat” their own internal components. Autophagy can be induced under nutrient deprivation or other stressful conditions. Thus, IRI was also noted to induce autophagy under certain circumstances. The purpose of this study was to imitate the clinical condition of SBS due to the mesenteric vascular leisions, to monitor the expression of autophagy and apoptosis after massive MBR(MBR), to explore the molecular mechanisms of intestinal adaption in which glucagon-like peptide 2(GLP-2) participated after MBR and to discuss the optimal time to receive the treatment of GLP-2 through the levels of autophagy and apoptosis fluctuation after the treatment at different time points postoperatively.Part Ⅰ. Expression of autophagy and apoptosis in the remnant bowel after MBRⅠ Objective The purpose of this section was to monitor the expression of autophagy and apoptosis at different time points postoperatively when compared with the sham-operated group.Ⅱ Methods(1) To imitate the clinical condition of SBS caused by IRI in animals, besides the animal models of sham operation and MBR only were also conducted;(2) To monitor the expression of autophagy at different time points postoperatively through combined methods of electron microscopy(EM), polymerase chain reaction(PCR)and Western Blotting, and a variety of autophagy markers including LC3, p62, Atg5 and Beclin-1;(3) To detect the expression of apoptosis through the method of Western Blotting by detecting the apoptosis-related protein PARP and Caspase 8.Ⅲ Results(1) The results of EM showed that the number of autophagic body within 24 hours after MBR was greater than the sham-operated group, and the number decreased significantly at postoperative 72 hours;(2) The expression of autophagy marker including LC3, Atg5 and Beclin-1 were significantly increased within postoperative 24 hours on the level of m RNA. Among them,the expression of Beclin-1 increased most significantly. These markers gradually decreased at postoperative 72 hours. Besides, the expression of P62 decreased within postoperative24 hours, while significantly increased at postoperative 72 hours in the experimental groups compared with the sham-operated group. There were siginificant differences among the groups of MBR only and IRI combined with MBR when compared with the sham-operated group(all p<0.01);(3) The results of Western Blotting also showed that the rate of transition from LC3Ⅰ to LC3 Ⅱ and the expression of LC3 Ⅱ was most significant within postoperative24 hours. Meanwhile the expression of Atg5 and Beclin-1 also heavily increased at these time points. Howerver, the expression of these markers gradually reduced after postoperative 72 hours. The expression of P62 didn’t change significantly within postoperative 24 hours, but increased significantly after postoperative 72 hours. There were siginificant differences among the groups of MBR only and IRI combined with MBR when compared with the sham-operated group(all p<0.001);(4) Meanwhile, the expression of apoptosis related protein PARP and Caspase 8significantly increased after postoperative 72 hours, while didn’t change much at postoperative 24 hours. There were siginificant differences among the groups of MBR only and IRI combined with MBR when compared with the sham-operated group(all p<0.001).Ⅳ Conclusion The expression of autophagy significantly increased within 24 hours after MBR while the expression of apoptosis didn’t, comparing with the sham-operated group. At postoperative 72 hours, the expression of autophagy gradually decreased while apoptosis increased substantially. Furthermore, the expression of autophagy and apoptosis was presumed to be stabilized at a basal level after postoperative 72 hours. It is also presumed that overexpression of autophagy initiates autophagic cell death within 24 hours after MBR,which functions similarly with apoptosis. Meanwhile, the expression of apoptosis didn’t change significantly at these time points. Decreased expression of autophagy seemed toplay a protective role at postoperative 72 hours when apoptosis was heavily induced. The increased apoptosis and decreased autophagy may increase self-renewal of enterocytes and promote the process of intestinal adaptation.Part Ⅱ. The molecular mechanism of autophagy involving in the treatment of GLP-2 after MBRⅠ Objective The purpose of this study was to explore the molecular mechamism of autophagy involving in the treatment of GLP-2 after MBR through the Akt-m TOR signaling pathway when compared with the groups receiving saline administration, and to detect the protective effects of GLP-2 on the intestinal mucosa injury after MBR.Ⅱ Methods:(1) Based on the methods in PartⅠ,GLP-2 and saline were received the same dose by introperitoneal injection for 3 days at different time points postoperatively in the group of IRI followed by MBR and MBR only, respectively;(2) The method of IHC was used to detect the expression of GLP-2R after the treatment of GLP-2 at different time points postoperatively;(3) The method of Western blotting was used to detect the expression of Akt-m TOR signaling moleculars including Akt, m TOR and 4EBP-1 after the treatment of GLP-2 at different time points postoperatively;(4) The methods of ELISA and HE staining were used to determine the level of LDH,an inflammatory cytokine, and to evaluate the degree of intestinal mucosa damage,respectively, after the treatment of GLP-2 at different time points postoperatively.Ⅲ Results(1) The results of IHC showed that the expression of GLP-2R increased significantly after the treatment of GLP-2 at postoperative 24 hours. However, the expression decreased after the treatment of GLP-2 at postoperative 72 hours. There were siginificant differences among the groups of MBR only and IRI combined with MBR after receiving the administration of GLP-2 when compared with the groups with normal saline administration( all p<0.001).(2) The results of Western blotting showed that the expression of Akt, m TOR and4EBP-1 increased initially at 16 hours postoperatively after the treatment of GLP-2, and reached to peak at 24 hours postoperatively, but decreased substantially at 72 hours postoperatively. There were siginificant differences among the groups of MBR only and IRI combined with MBR after receiving the administration of GLP-2 when compared with the groups with normal saline administration(all p<0.001).(3) The results of ELISA revealed that the treatment of GLP-2 didn’t decrease the value of LDH significantly(p=0.282).(4) The results of HE staining revealed that the treatment of GLP-2 could alleviate the intestinal mucosa injury at different time points postoperatively(all p<0.001).ⅣConclusion The expression of Akt-m TOR signaling moleculars initiated to increase significantly at16 hours postoperatively after the treatment of GLP-2, comparing with the group with the treatment of saline, and reached to peak at 24 hours postoperatively. It was noted that GLP-2 could activate Akt-m TOR signaling pathway through binding with GLP-2R after treatment at postoperative 24 hours. Consequently, activation of m TOR could lead to significant inhibition of autophagy which proved to participant in the process of intestinal adaption after MBR. It was also speculated that PI3 K Ⅲ-beclin1 and other autophagy-related signaling pathways may be involved in these processes. Moreover, the results also revealed that the treatment of GLP-2 couldn’t reduce the level of intestinal inflammation but could significantly alleviate the intestinal mucosal damage.Part Ⅲ. The optimal time to receive the treatment of GLP-2 for early intestinal rehabilitation after MBRⅠ Objective The purpose of this study was to explore the optimal time to receive GLP-2administration for early intestinal rehabilitation after MBR through the levels of autophagy and apoptosis fluctuation when compared with the groups receiving saline administration.Ⅱ Methods(1) The methods of animal models(see PartⅡ);(2) The methods to detect the expression of autophagy and apoptosis(see PartⅠ).Besides, the method of IHC and TUNEL were also used to detect the expression of autophagy and apoptosos in this part, respectively.Ⅲ Results(1) The results of EM showed that the number of autophagic body was significantly reduced after the treatment of GLP-2 at postoperative 24 hours when compared with the groups receiving saline administration;(2) The expression of autophagy marker including LC3, Atg5 and Beclin-1 was heavily reduced after the treatment of GLP-2 at postoperative 24 hours on the level of m RNA. Among them, the level of Beclin-1 was the most significantly reduced. While the expression of P62 significantly increased after the treatment at this time point. There were siginificant differences among the groups of MBR only and IRI combined with MBR after receiving the administration of GLP-2 when compared with the group receiving normal saline administration( all p<0.001);(3) The results of Western Blotting showed that the rate of transition from LC3 Ⅰto LC3 Ⅱ and the expression of LC3 Ⅱ were reduced significantly after the treatment of GLP-2 at postoperative 24 hours. Meanwhile, the levels of Atg5 and Beclin-1 were also reduced at this time point. However, the levels of P62 decreased significantly after the treatment. Furthermore, the expression of autophagy increased significantly after the treatment of GLP-2 at postoperative 72 hours. There were siginificant differences among the groups of MBR only and IRI combined with MBR after receiving the administration of GLP-2 when compared with the group receiving normal saline administration( all p<0.001);(4)The expression of apoptosis related proteins PAPR and Caspase 8 heavily increased after the treatment of GLP-2 at postoperative 72 hours, while didn’t change much at postoperative 24 hours. Moreover, the expression of apoptosis increased substantially after the treatment of GLP-2 at postoperative 7 days. There were siginificant differences among the groups of MBR only and IRI combined with MBR after receiving the administration of GLP-2 when compared with the groups with normal saline administration( all p<0.001);(5) The results of IHC also revealed that the expression of LC3, Atg5 and Beclin-1was reduced after the treatment of GLP-2 at postoperative 24 hours. While the expression of P62 increased significantly at this time point. Besides, the expression of autophagyincreased heavily after the treatment of GLP-2 at postoperative 72 hours. There were siginificant differences among the groups of MBR only and IRI combined with MBR after receiving the administration of GLP-2 when compared with the groups with normal saline administration( all p<0.01);(6) The results of TUNEL showed that the number of autophagic body was significantly reduced after the treatment of GLP-2 at postoperative 72 hours. While the number increased significantly at postoperative 7 days. There were siginificant differences among the groups of MBR only and IRI combined with MBR after receiving the administration of GLP-2 when compared with the groups with normal saline administration( all p<0.001).Ⅳ Conclusion The expression of autophagy and apoptosis fluctuation wasn’t significant at postoperative 8 hours and 16 hours after the treatment of GLP-2, comparing with the groups receiving the treatment of saline. However, the expression of autophagy significantly decreased at postoperative 24 hours after the treatment of GLP-2, while the expression of apoptosis didn’t change siginificantly. It was also noted that 3 days of GLP-2administration initiated at postoperative 72 hours heavily inhibited the expression of apoptosis. Based on the results in this section, it is presumed that the treatment of GLP-2initiated at postoperative 24 hours is the optimal time for early intestinal rehabilitation after MBR. Further studies regarding the long-time effect of this treatment needs to be investigated in the future.