The Impact Of A Newly Adipokine Apelin On Crohn’s Disease Based On Its Effects On Intestinal Lymphatic Vessels

Crohn’s disease(CD)is a chronic and recurrent inflammatory bowel disease.The etiology of CD is currently unknown,and it is commonly accepted that inappropriate interactions between intestinal pathogens and host immunity in genetically susceptible individuals may contribute to the basic pathophysiology of CD.Dysfunction of intestinal lymphatic vessels is a common histological change which may answer for the mucosal edema and intestinal inflammation.Decreased lymphatic vessel density was found to be related to the postoperative recurrence of CD patients.Amelioration of the intestinal inflammation by promoting the lymphatic vessels in animal colitis models indicates that strategies targeting lymphatics may be a new method for CD treatment.Thickening mesentery and creeping fat have been particularly recognized since this disease was first described,which helped surgeons to distinguish the diseased mesentery from normal mesentery.Emerging evidence strongly suggests that the morphologic and/or functional abnormalities of mesenteric adipose tissue(MAT)may play a potential role in the pathogenesis and disease progression of CD.Adipokines,regarded as produced predominantly by adipocytes,have been described to exert distinguishing effects on both metabolism and the immune system.Altered production of adipokines in the MAT of Crohn’s patients,such as adiponectin,leptin,demonstrates a strong correlation with their capacity to modulate the immune response and even the microenvironment within the adipose tissue.Apelin is a newly identified adipokine that produced and secreted by adipocytes.Studies reported that apelin would be involved in the modulation of the cardiovascular system,fluid homeostasis and energy metabolism,but limited researches focused on its role in CD.Advanced studies have revealed the essential role of apelin during the lymphatic development in zebrafish,and apelin has also been shown to be critical for the progress of proliferation and migration of the lymphatic endothelial cells in vitro and flow-induced endothelial cell polarization.Further studies demonstrated that apelin enhances the integrity of lymphatic vessels and attenuates edema formation and inflammation by promoting lymphatic function in vivo.However,whether apelin has a potential role in the lymphatic vessel in CD status remains exploration.This study firstly investigated the expression of Apelin,the new adipokine,in the serum and mesenteric adipose tissue of CD.Based on the results,we chose Il-10-/-mice as our animal model of CD and explored whether apelin could ameliorate the inflammation of Il-10-/-mice.Then we focused on the intestinal lymphatics,aiming at finding out the specific role of Apelin.The study was divided into two sections:Part Ⅰ The expression of Apelin in serum and mesenteric adipose tissue of patients with Crohn’s diseaseObjective:To clarify the expression of Apelin in serum and mesenteric adipose tissue of CD patients and related factors.Methods:101 cases of CD and 67 outpatients were included.ELISA was used to detect the expression of Apelin in the serum of the two groups.The relationships between the Apelin concentration and the clinical data of CD patients were also analyzed.Twenty-four CD patients and 12 non-CD patients were included to detect the expression of Apelin in mesenteric adipose tissue by immunohistochemistry.The expression of Apelin in normal adipose tissue and hypertrophic adipose tissue was also compared in 10 CD patients.In Il-10-/-mice and wild-type mice,the expression of Apelin in mesenteric adipose tissue of two groups of mice was compared.Results:The serum Apelin concentration in the CD group was significantly higher than that in the Control group(621.2 ng/ml,IQR 391.1-1596.3 ng/ml vs 239.2 ng/ml,IQR 127.4-320 ng/ml).The high expression of Apelin in the CD group correlated with mesenteric fat index(R2=0.71,p<0.05).Apelin concentration in active CD patients was higher than that in remission patients(882.5ng/ml,IQR 221.8-1186ng/ml vs 442.7ng/ml,IQR 201.7-481.7ng/ml,p<0.05),In patients with CRP≥10mg/L,Apelin concentration was higher than that in patients with CRP<10 mg/L(1147 ng/ml,IQR 245.4-2083 ng/ml vs 657.7 ng/ml,IQR 178.7-486.8 ng/ml,p<0.05).The expression of Apelin in mesenteric adipose tissue in CD group was significantly higher than that in Control group(relative IOD:9.04 ± 3.19 versus 1.00 ± 0.51,p<0.05),and the expression of Apelin in htMAT group was significantly higher than that in nMAT group(relative IOD:1.9±0.23 versus 1.00±0.22,p<0.05).Apelin was also significantly higher in mesenteric adipose tissue of Il-10-/-mice than in wild-type mice(relative IOD:0.31±0.05 versus 0.20±0.02,p<0.05).Conclusion:Serum Apelin concentration in CD patients is higher than normal people.High level of Apelin is associated with visceral adipose and disease activity.Apelin expression in mesenteric adipose tissue of CD patients and model mice was increased compared with the control group.Part Ⅱ Density Detection and Morphological Observation of Intestinal Lymphatic Vessels in Crohn’s DiseaseObjective:To compare the density of intestinal lymphatic vessels in CD patients and non-CD patients and observe the morphological structure of lymphatic vessels.Methods:Twenty-four CD patients were enrolled as CD group,and 12 non-CD patients were enrolled as control group.Intestinal lymphatic vessel density was detected by immunohistochemistry in two groups and the morphology of lymphatic vessels was observed by H&E staining.Results:The lymphatic vessel density was significantly higher in CD group then in Control group(12.07%;IQR,10.45-13.73%versus 5.41%,IQR,4.30-6.65%,p<0.05).Abnormal morphology of lymphatic vessels was found in CD group,such as lymphatic obstruction,perilymphangitis and occlusive granuloma.Conclusion:Lymphatic vessel density was increased in CD patients,with the presence of lymphatic obstruction,perilymphangitis and occlusive granuloma.Part Ⅲ Impact of Apelin on the intestinal inflammation of Il-10-/-mice and its specific role in lymphatic vesselsObjective:To determine the effect of apelin on intestinal inflammation in Il-10-/-mice and its effect on intestinal lymphatic vessels.Methods:The 15-week-old wild-type mice were selected as WT group,15-week-old Il-10-/-mice were randomly divided into Control group and Apelin group.Mice in Apelin group were given intraperitoneal injection of 0.1umol/kg Apelin every day.WT group and control were given the same dosage of normal saline.The weight was recorded weekly and Disease Activity Index(DAI)of each mouse was evaluated,and the drug was executed 4 weeks later.Intestinal inflammation and inflammatory cytokines in three groups was analyzed by H&E staining and q-PCR technique.The intestinal lymphatic density in three groups of mice was compared by immunohistochemical analysis and western-blot technique.The function of intestinal lymphatic drainage was evaluated by Evans Blue dye and fluorescent lymphangiography.Finally,the Western blot technique was used to analyze the expression of related signaling pathways.Results:Systemic delivery of apelin significantly ameliorated chronic colitis in Il-10-/-mice,demonstrated by decreased disease activity index and inflammatory scores,and lower levels of Tnf-α,Il-1β and Il-6(p<0.05).Increased LV density and podoplanin levels indicated that apelin promoted lymphangiogenesis.Evans blue dye and fluorescent lymphangiography revealed an enhanced lymphatic drainage function in apelin-treated mice.The role of apelin was found to be related to the activation of the Akt and Erk signaling pathways(p<0.05).VEGFR3 revealed comparable expression among three groups(p>0.05).Compared with WT group,the expression of VEGF-C protein both in control group and Apelin group increased,but there was no difference between the two group(p>0.05).Conclusion:Apelin can significantly reduce intestinal inflammation in Il-10-/-mice,and the improvement of inflammation may be achieved by increasing intestinal lymphatic density and promoting intestinal lymphatic drainage function.The effect of Apelin on lymphatic vessels is related to the activation of Akt/Erk signaling pathway,and independent on the VEGF-C/VEGFR3 signaling pathway.