Experimental Study On Prevention Of EMT By C5aRA And Improvement Of Abdominal Adhesion

Ⅰ.Experimental study on the protective effect of C5 a RA on damaged rat peritoneal mesothelial cells(RPMCs)Objective To investigate the protective effect of C5 a RA on damaged RPMCs.Methods 1 The peritoneal mesothelial cells of SD rats were extracted by enzymatic hydrolysis.2 CCK-8 was used to detect the activity of RPMCs.3.To study the protective effect of C5 a RA on damaged RPMCs.Control group,LPS group(20μg/ m L,50μg/ m L,100μg/ m L),C5 a RA+LPS group(100μg/ml LPS +5μg/ml C5 a RA).The morphological changes of cells in each group were observed under fluorescence microscope after HE staining and ghost cyclic peptide staining.Results 1.The extracted RPMCs were polygonal,closely arranged,and pebble-like in appearance.2 CCK-8 results showed that LPS decreased the activity of RPMCs in a concentration-dependent manner.C5 ARA can effectively protect the proliferative activity of damaged cells.3.Morphological results showed that the cells in the LPS group were oval,while the cells in the C5ARA+LPS group were more similar to those in the Control group,and the density was significantly increased.Conclusion C5 a RAs can reduce the damage of RPMCs caused by LPS.Ⅱ.Experimental study on inhibiting EMT of human peritoneal mesothelial cells by C5 a R Objective To investigate the effect of C5 a RA on TGF-β1-induced EMT in HPMCs and its mechanism.Methods 1 Cell morphology:(1)Control group(2)TGF-β1 group(10ng/ml TGF-β1)(3)C5ARA+TGF-β1 group(5μg/ml C5 a RA +10ng/ml TGF-β1).After 6h and 12 h treatment,the morphology and density of HPMCs were observed under an inverted microscope.The cytoskeleton of HPMCS was observed under confocal laser microscope by staining the HPMCS microfilament with cyclopeptides.2 Immunofluorescence staining was used to detect the expression of E-cadherinα-SMA in each group,and the experimental grouping was consistent with the cell morphology.Results 1 Morphological changes of TGF-β1 cells: Under light microscope and confocal laser microscope,TGF-β1 cells were mostly fusiform compared with the Control group;The cell morphology of the C5 Ar +TGF-β1 group was more similar to that of the Control group.2Expression of EMT-related proteins: Under the action of TGF-β1,the expression of Ecadherin in HPMCs was decreased,and the expression of α-SMA was increased.Compared with TGF-β1 group,E-cadherin expression in HPMCs was up-regulated and α-SMA expression was down-regulated after administration of C5 a RA.Conclusion C5 a RA can alleviate EMTⅢ.Experimental study on the improvement of abdominal adhesion in mice by inhibiting EMT with C5 a RObjective 1 Immunohistochemical staining was used to determine the changes of neutrophils expression in the adhesion sites of mice after operation.2 To evaluate the effect of C5 a RA on the improvement of postoperative abdominal adhesion in mice.3 The expression changes of α-SMA,TGF-β1 and OPN were detected by immunofluorescence technique,and the mechanism of C5 a RA to improve the occurrence of postoperative abdominal adhesion in mice was studied.Methods Seventy-five Kunming mice were randomly divided into three groups:(1)sham operation group(open abdominal cavity to expose intestinal wall and suture),(2)model group(mechanical injury method to prepare abdominal adhesion model),(3)C5aRA group(mechanical injury method to prepare abdominal adhesion model).On the 1st,3rd and 5th day after operation,5 animals in each group were sacrificed to observe the expression of neutrophils in the injured site by immunohistochemical staining.On the 7th and 14 th days after surgery,5 animals in each group were sacrificed for necropsy and the adhesion of mice was graded.He,Masson and Sirius red staining techniques were used to evaluate the adhesion area,inflammation and fibrosis of the adhesive tissue under the microscope.The expression of EMT-related proteins α-SMA,TGF-β1 and OPN in the adhesive tissue was observed by immunofluorescence staining.Results 1 The expression of neutrophils in the sham operation group was negative at day1,3 and 5,while the nuclei and cytoplasm of neutrophils in the model group were strongly positive and the number of neutrophils was large.Compared with the operation group,the positive expression of neutrophils in the C5 a RA group was significantly reduced.2 No adhesion occurred in the sham operation group.The intensity,range,tissue inflammation and degree of fibrosis of postoperative adhesion in the C5 a RA group were lower than those in the model group.3 Compared with sham operation,the expression of α-SMA,TGF-β1 and OPN in model group were up-regulated;After treatment with C5 AR,the expressions of α-SMA,TGF-β1 and OPN in the C5 a RA group were down-regulated compared with those in the model group.Conclusion C5 a RA has a certain anti-adhesion effect,the mechanism may be related to the inhibition of inflammatory exudation and EMT by C5 AR.Figure 16;Table 1;120 articles...