Abstract:

Objective To investigate the ameliorative effect of the injectable oxidized dextran（ODex）/carboxymethyl chitosan（CMCS） hydrogel （DCC） loaded with exosomes（Exo） （DCC/Exo） on postoperative peritoneal adhesion （PA） in the mice，and to elucidate its mechanism. Methods The exosomes were extracted from the rats， and DCC/Exo was prepared and its chemical structure was characterized. Immunohistochemical staining method was used to detect the expression of the inflammatory factor Ly-6G in mouse dorsal skin tissue after injection of DCC/Exo to evaluate its biocompatibility. The mouse PA model was established by cecal abrasion. The KM mice were randomly divided into control group， hyaluronic acid （HA） group， DCC group， and DCC/Exo group. The PA condition was observed and scored in the mice in various groups at 5 and 14 d after operation. HE staining was used to observe the pathomorphology of PA tissue of the mice in various groups. At 5 d after operation， immunohistochemical staining method was used to detect the expression levels of myeloperoxidase （MPO） in PA tissue of the mice in various groups. The dihydroethidium （DHE） fluorescence probe assay was used to detect the levels of reactive oxygen species （ROS） in PA tissue of the mice in various groups； immunofluorescence staining was used to detect the expression levels of the oxidative damage marker 8-hydroxy-2'-deoxyguanosine （8-OHdG） and the mesothelial-mesenchymal transition （MMT）- related proteins E-cadherin and Vimentin in PA tissue of the mice in various groups. The Mitotracker fluorescence probe assay was used to detect the degree of mitochondrial damage in PA tissues of the mice in various groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） staining was used to detect the apoptotic rates of the cells in PA tissue of the mice in various groups. Results The isolated Exos exhibited a cup-shaped double-membrane structure. After loading with Exos， the characteristic absorption peaks of the DCC hydrogel showed no significant shift. The immunohistochemistry analysis results revealed only minimal neutrophil infiltration in mouse dorsal skin tissues. At 5 and 14 d after operation， compared with control group and DCC group， the mean adhesion score of the mice in DCC/Exo group was significantly decreased （P<0.001）. The HE staining results showed severe PA with massive inflammatory cell infiltration of the mice in control group， mitigated PA in the mice in HA group and DCC group， and no adhesion with only a few inflammatory cells observed in the abdominal cavity of mice in DCC/Exo group. At 5 d after operation， the immunohistochemical staining results showed significant diffuse inflammatory infiltration in PA tissue of the mice in control group. Compared with control group and DCC group， the expression level of MPO in PA tissue of the mice in DCC/Exo group was significantly decreased （P<0.001）. The DHE staining results showed that compared with control group and DCC group， the ROS level in PA tissue of the mice in DCC/Exo group was significantly decreased （P<0.001）.The immunofluorescence staining results showed that compared with control group and DCC group， the expression levels of 8-OHdG and Vimentin in PA tissue of the mice in DCC/Exo group were significantly decreased （P<0.001）， while the expression level of E-cadherin was significantly increased （P<0.001）. Compared with control group and DCC group， the fraction of Mitotracker fluorescence area in PA tissue of the mice in DCC/Exo group was significantly increased （P<0.001）， indicating a decreased degree of mitochondrial damage. Compared with control group and DCC group， the apoptotic rate of the cells in PA tissue of the mice in DCC/Exo group was significantly decreased （P<0.001）. Conclusion Topical application of the DCC/Exo composite hydrogel may effectively attenuate postoperative PA in the mice， and its mechanism may be related to the suppression of local inflammatory infiltration， reduction of tissue oxidative stress， decrease in apoptosis， and inhibition of the MMT process.

Key words: Postoperative peritoneal adhesion, Hydrogel, Exosome, Oxidative stress, Mesothelial-mesenchymal transition