Research On The Amelioration Of Gastrointestinal And Brain Injury Induced By MC4R Agonist After The Secondary Intra-abdominal Hypertension

Background: Severe exsanguinating hemorrhage, burning, abdominal trauma are often likely to induce the intra-abdominal hypertension(IAH), even abdominal compartment syndrome( ACS) during the medical intervention, which commonly involves in multiple intra-abdominal organs, such as gastrointestinal track, liver and kidney, as well as some remote organs, like brain, heart and lung. The classification of IAH/ACS refers to the primary, secondary and recurrent. The primary IAH/ACS is caused by abdominal/pelvic injuries; the secondary would more likely to be caused by other general critical conditions, such as Systemic Inflammatory Response Syndrome(SIRS), shock and severe burns; the recurrent IAH/ACS refers to the condition in which IAH or ACS redevelops following previous ones being treated with surgery or drugs. The risky factors of IAH include the massive fluid transfusion, the capillary leak, the increased amount of intra-abdominal volume and the decrease of abdominal wall compliance, in which the posttraumatic massive fluid therapy is the primary risk factor. The pathology and etiology of IAH has not been clear yet. Currently, the approaches to IAH mostly refer to the decompression by laparotomy. However, there are still several concerns such as the ischemic-reperfusion lesion in the aftermath of decompression. As the edema of gastrointestinal(GI) track is the main cause of increase of abdominal pressure, this research aimed to the amelioration of edema GI track as well as the lesion of brain, the extra-abdominal vital organ.Recently, more and more interests were in the melanocortin(MC), which mediates the chromatosis, the energy hemostasis, inflammatory response and immunological regulation in multiple organs via receptors. Studies has referred the synthetic analogs play roles protecting the body against various diseases, such as hemorrhagic shock, intestinal ischemia, ischemic stroke, and traumatic brain injury as well as the prompt anti-inflammatory function bypass the cholinergic anti-inflammatory pathway, whose mechanism remains unclear.This goal of this research is to explore the effects of MC on dysfunctional mid-gut and brain associated with the secondary IAH and the further mechanism with regards to initiate novel strategies to treat the patients sustaining ACS.The animal experimental model with the secondary IAH was set up to verify this hypothesis, which is practical, repeatable model to simulate the etiology of IAH. Based on the model, the effects of the melanocortin 4 receptor agonist RO27-3225 in reducing the lesion of gut induced by IAH, and the related mechanism as well as in protecting function the rats’ IAH brain injuries were observed. The regulatory effect of cholinergic pathway was studied.Methods1. Animal model. The secondary IAH model was set up with the different mean arterial pressure(MAP) caused by femoral artery bleeding after intubation :40mm Hg for 120 min, 30 mm Hg for 90 min and 25 mm Hg for 60 min followed by sustained IAP of 20 mm Hg for 4 hours. Wet/dry ratio was used to analyze the tissue water in the GI track and brain, HE dying was employed to assess the morphology of GI track, and the survival rate was also analyzed. By comparing between the groups, the applicable model with both acceptable survival rate and the IAH was identified for the following study.2. In the secondary IAH model(30mm Hg for 90 min followed by sustained IAP of 20 mm Hg for 4 hours), the MAP was continuously monitored, the real-time PCR was applied to assess the inflammatory cytokines(TNF-α and IL-1β), the tissue water was determined by the wet/dry ratio, HE dye was used to morphologically tissue lesion, intestinal fatty acid-binding protein(I-FABP) concentrations were measured by using enzyme-linked immunosorbent assay(ELISA), intestinal permeability was measured and the effect of the key factors, Rho-associated coiledecoil-containing protein kinase 1(ROCK1), phosphorylated myosin light chain expression and SOD activity were evaluated by western blot assay. Based on those tests, the protective effect of RO27-3225 was studied.3. In the secondary IAH model, the water content of brain was also represented by wet/dry ratio, the Evans Blue(EB) dye extravasation method was used to detect changes in the permeability of the blood brain barrier(BBB), the real-time PCR and ELISA was used to analyze the cerebral inflammatory cytokines,(TNF-αand IL-1β), as well as the aquaporin4(AQP4) and matrix metalloproteinase 9(MMP9) levels. The protective effect of the RO27-3225 was also analyzed based on relative experiment.Results:1. The model with precondition of MAP 30 mm Hg for 90 minutes followed by sustained IAP of 20 mm Hg for 4 hours was applicable for further study, which was able to induce significant lesions in the gastrointestinal(GI) track with desirable survival rate.2. RO27-3225 restored mean arterial pressure, reduced tumor necrosis factor-a, and interleukin-1b messenger RNA expression which was increased by IAH, alleviated histologic damage, levels of ROCK1 and phosphorylated myosin light chain expression, decreased intestinal fatty acid-binding protein(I-FABP), GI edema, permeability and morphological lesions. The protective effects would be reversed by the specific antagonist HS024 and nicotinic acetylcholine receptor antagonist Chl diiodide.3. The effects of the melanocortin 4 receptor agonist RO27-3225 induced the alleviation of the rats’ IAH cerebral edema, down-regulate the expressions of the IL-1β and TNF-α inflammatory cytokines, the AQP4 and MMP9 levels, which resulted in the alleviation of brain damage. The specific antagonist HS024 and nicotinic acetylcholine receptor antagonist Chl diiodide would abrogated the protective effects of RO27-3225, however, no significant effects were showed in the proliferation of neuroglia cellsConclusions:1. IAH could induce both intra-abdominal-GI track lesions which included edema and inflammatory response and extra- abdominal organ-brain damage which referred to the cerebral edema, inflammation and the disintegrity of BBB.2. Selective MC4 receptor agonist could stablize hemodynamics by reversing the intestinal permeability via regulating the expression of ROCK1 and phosphorylated myosin light chain, and counteracts the intestinal inflammatory response to ameliorate the intestinal injury in experimental secondary IAH The protective effect may be through the cholinergic pathway.3. Selective MC4 receptor agonist could effectively ameliorate the disintegrity of BBB by decrease the expression of MMP 9, relieve the cerebral edema by regulating AQP4, as well as reduce the inflammatory response, which significantly compromised the brain lesions caused by IAH. The protective effect may be through the cholinergic pathway. However, no significant effect was observed on the quantity of neuroglia cells in the acute phase.