| Objective During sepsis,the body has an uncontrolled inflammatory response to the infection,a large number of inflammatory factors are released,multiple inflammation-related signaling pathways are activated,and finally multiple organ dysfunction occurs,the mortality rate is high,and there are few treatment measures and poor results.Expensive.There is an urgent need to find new treatment entry points.The intestine is the body's largest bacterial pool.Excessive inflammatory reactions affect the intestine during sepsis,and the intestinal mucosal barrier is damaged.A large number of bacteria and endotoxins enter the bloodstream,aggravating the inflammatory response,and promoting the process of subsequent organ damage.Therefore,the intestinal tract is not only a damaged organ of sepsis,but also an important intermediate link in the development of sepsis into multiple organ dysfunction,so the protection of the intestinal barrier function in sepsis is very important.This experiment investigated the effect of bone marrow mesenchymal stem cell exosomes on the intestinal barrier function of sepsis rats and explored its related mechanism.The purpose is to provide a method and theoretical basis for the protection of intestinal barrier function in sepsis.MethodsPart I Extraction of bone marrow mesenchymal stem cells and exosomes: experimentally extract bone marrow mesenchymal stem cells from rat tibia and femur,isolate and culture primary cells using the whole bone marrow adherent method,digest and culture passage cells,and use light microscopy observe the cell morphology,and use cell cytometry to detect cell surface antigens for identification;extract the exosomes of bone marrow mesenchymal stem cells by centrifugation,observe the morphology of exosomes under electron microscope,and use surface cytometry to detect surface markers for identification.Preserve exosomes for subsequent experiments.Part II Modeling and survival rate observation: 18 rats were randomly divided into 3 groups:(1)normal control group(n = 6)(2)sham operation group(n = 6),laparotomy was performed after anesthesia,and abdomen was closed layer by layer after turning the cecum,rats were injected with physiological saline 20 m L/kg subcutaneously to prevent shock.(3)cecal ligation and puncture caused sepsis(CLP)group(n = 6).CLP was used to prepare rat model of sepsis group.Rats were injected with physiological saline 20 m L/kg subcutaneously to prevent shock.Observe the general condition,abdominal condition and survival rate of rats after operation.Part III Effect of bone marrow mesenchymal stem cell exosomes on intestinal barrier function in sepsis rats and discussion of related mechanisms: 48 rats were randomly divided into 4 groups,and the specific group was(1)sham operation + normal saline group(n = 12): Turn the cecum and close the abdomen after suture,and suture the abdominal wall,and inject 100 microliters of normal saline through the tail vein 2 hours after surgery;(2)cecal ligation and puncture caused sepsis and normal saline(CLP + normal saline)group(n = 12):Rats underwent CLP and were injected with 100 microliters of normal saline via tail vein 2 hours after surgery;(3)cecal ligation and puncture caused sepsis+exosomes(CLP+exosomes)group(n = 12): Rats underwent CLP,and 100?l of a diluted solution containing 30?l of mesenchymal stem cell exosomes was injected through the tail vein 2 hours after the operation;(4)cecal ligation and puncture caused sepsis + exosome-free supernatant(CLP + exosome-free supernatant)group(n=12): Rats underwent CLP,and 100 microliters of the exosome-free supernatant was injected through the tail vein 2 hours after surgery.At six time points and 24 hours,6 rats were taken at each time point.Plasma and small intestine tissues were taken.Pathological changes in intestinal tissue were observed under a light microscope.Detection of D-lactic acid,diamine oxidase(DAO),endotoxin levels in plasma and tumor necrosis factor-a(TNF-a),interleukin-6(IL-6)and myeloperoxidase(MPO)level in intestinal tissues by ELISA,reflecting intestinal permeability and inflammatory changes during sepsis,and the effects of bone marrow mesenchymal stem cell exosomes on them;Westernblot was used to detect MAPK signaling pathway and NF-kB B signaling pathway phosphorylation in sepsis rats of different groups to explore the mechanism of the effect of bone marrow mesenchymal stem cells exosomes on intestinal barrier function in sepsis.ResultsPart I Extraction of bone marrow mesenchymal stem cells and exosomes: 24 hours after inoculation of bone marrow mesenchymal stem cells,some cells adhered,and the cells changed from polymorphic to spindle-shaped after 3-4 days,and vortex colonies appeared after 1 week.After 9-10 days,the cells gradually fused into pieces.After digestion and passage for 24 hours,the cells were completely adherent,vigorously grown,and uniformly shaped like a spindle.Flow cytometry showed that the cultured bone marrow mesenchymal stem cells uniformly expressed CD44,CD105(> 95%),CD34,CD45(<2%).Bone marrow mesenchymal stem cell exosomes were isolated by centrifugation,and vesicle-like structures were observed under electron microscope,the size of which was about 30-60 nm.Flow cytometry showed that the surface markers CD9,CD63,and CD81 were expressed.Part II Modeling and survival rate observation: CLP was used to produce a sepsis rat model.The rat's morbidity gradually became apparent about 6 hours after operation,and symptoms such as atrophy,shortness of breath,reduced eating and drinking water,and eruption were observed.Abdominal cavity was observed.It was found that in the CLP group,the bowel arrangement was disordered in the abdominal cavity 6 hours after CLP,the cecum was swollen and dilated,and a small amount of effusion was seen in the abdominal cavity.With time,the intestinal swell and expansion obviously showed adhesion and necrosis,and the peritoneal effusion gradually increased.Within 72 hours,no death was found in the sham operation group,and the survival rate in the CLP group was 50%,and the death time of the rats was mainly concentrated within 24-48 hours after the operation.Part III Effects of bone marrow mesenchymal stem cell exosomes on intestinal barrier function in sepsis rats and the related mechanisms: at 6 and 24 hours,the small intestine villi in the sham operation + normal saline group were neatly arranged under light microscope,The small intestinal mucosa structure is basically complete.The small intestine villi are sparsely arranged in the CLP + saline group and the CLP + exosome-free supernatant group.Some of the villus and lamina propria fall off,capillaries dilate,inflammatory cells infiltrate,and CLP + exosomes group mucosal congestion and edema,no villi and lamina propria detachment,Chius score found that the score of rat intestinal tissues in the CLP + saline group and CLP + exosome-free supernatant group was significantly higher than that in the sham operation + saline group(P <0.05),while the CLP + exosomes group was significantly lower than the CLP + saline group and CLP + exosome-free supernatant group(P <0.05);at the two time points of 6 hours and 24 hours,Compared with the sham operation + normal saline group,the plasma levels of D-lactic acid,DAO and endotoxin in the CLP + normal saline group and the CLP + exosome-free supernatant group were significantly increased(P <0.05),while the CLP + exosomes group compared with the CLP + normal saline group and the CLP + exosome-free supernatant group,plasma D-lactic acid,DAO,and endotoxin levels were significantly reduced(P <0.05);compared with the sham operation + normal saline group,the CLP + normal saline group and the CLP + exosome-free supernatant group the levels of TNF-?,IL-6 and MPO in the intestinal tissues of rats were significantly increased(P <0.05),while the CLP+exosomes group,compared with the CLP+normal saline group and the CLP + exosome-free supernatant group,the levels of TNF-?,IL-6 and MPO in the intestinal tissues were significantly reduced(P <0.05);compared with the sham operation + normal saline group,the CLP + normal saline group and the CLP + exosome-free supernatant group,the levels of phosphorylation of ERK,P38,JNK and NF-kB in the intestine tissues were significantly increased(P <0.05),while the CLP+exosomes group compared with the CLP+normal saline group and the CLP+ exosoma-free supernatant group,the levels of phosphorylation of ERK,P38,JNK and NF-kB in the intestinal tissues were significantly reduced(P <0.05).Conclusions 1Bone marrow adherent method can successfully isolate and culture bone marrow mesenchymal stem cells,and centrifugation method can successfully extract exosomes.2 Cecal ligation and puncture can successfully prepare a sepsis rat model.3 Bone marrow mesenchymal stem cell exosomes can inhibit intestinal inflammation in sepsis rats,reduce intestinal mucosal permeability and protect the intestinal barrier function.4 Bone marrow mesenchymal stem cell exosomes can protect the intestinal barrier function by inhibiting the activation of MAPK signaling pathway and NF-?B signaling pathway in the intestinal tissue of sepsis rats.. |
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