Study On The Effects Of Adipose Mesenchymal Stem Cell Exosomes On Inflammation Regulation In Sepsis Rats

Objective（s）:To investigate the effects of adipose derived stem/stromal cells-exosomes（adipose derived stem/stromal cells-exosomes,ADSCS-exo）derived from SD（Sprague Dawley,SD）rats on inflammation regulation in sepsis models established using cecal ligation and puncture（CLP）.Methods:Fat pad tissue from the groin on both sides of SD rats is taken in a sterile environment,adipose derived stem/stromal cells（ADSCs）are cultivated and amplified in vitro using a tissue block attachment method,ADSCs are induced by an inducing differentiation medium when cultured to the P₃generation,detecting their ability to differentiate in multiple directions in lipogenesis and osteogenesis,and detecting the expression of specific markers on their surface using flow cell technology.The supernatant solution cultured by ADSCs was collected,ADSCs-exo was extracted by differential centrifugation,and the shape,size,and structure of ADSCs-exo was observed by transmission electron microscopy;nanoparticle tracking analysis（NTA）detected the concentration and particle diameter distribution range of the original solution;protein blotting experiments detected the expression of surface markers CD9 and CD63 and identified them as ADSCS-exo.Fifty-six SPF grade SD rats,female,130 to 150g,were randomly divided into 3 groups,namely:sham surgery group（Sham）,sepsis group（CLP）,and exosome group（CLP+ADSCs-exo）.CLP surgery modeled sepsis in SD rats.3h after surgery,the exosome group injected 400ug of ADSCS-exo into the abdominal cavity（1 ug/ul for measuring exosome protein concentration）.The total injection volume was about 400 ul.The Sham group and CLP group were given an intraperitoneal injection of the same volume of 1×PBS400ul.Observe behavioral changes in each group of rats,count survival rates,and draw a 72h survival curve.Blood was collected from the lower abdominal aorta 48hours after modeling,enzyme linked immunosorbent assay（ELISA）detection the expression of inflammatory factors such as TNF-α,IL-6,IL-1β,IL-10;collection of HE staining of histopathological sections of liver,kidney,and small intestine tissues to compare damage and inflammation infiltration in various groups of liver,kidney,and small intestine;real-time quantitative polymerase chain reaction（q PCR）and protein blot（western blot）to detect TNF-α,IL-6,IL-1β,IL-4,IL-10 in various groups of small intestine tissue level of expression of m RNA and proteins.Results:1.ADSCs culture and identification results:（1）After 48 hours of culturing primary ADSCs,a small number of short spindle-shaped cells climbed out of the periphery of the tissue block.After 4-5 days,they swirled around the tissue mass,grew and spread like a fish group,and were transmitted around 8-9d.After transmission,the P₃and P₄generation ADSCs were all long-spindle-shaped and spindle-shaped,and grew in a vortex and fiber-like arrangement.There was no obvious widening and differentiation of the cicada wings.（2）Flow cytometry detected that the P₃generation ADSCs surface markers CD44 and CD90 were positive,and CD34 and CD45 were negative.After induction of lipogenesis,oil red O staining showed dark red round lipid droplets of varying sizes within cells.After induction of osteogenesis,alizin red staining showed irregularly shaped calcified nodules of orange-red color within the cells.2.Identification results of ADSCs-exo:（1）A round or oval bilayer membrane vesicle of a uniform size is seen under a transmission electron microscope.The low electron density region can be seen in the center of the vesicle with a diameter of about 130nm,which is in line with the external characteristics of the mesenchymal stem cell exosome.（2）The NTA results showed that the particle concentration in the undiluted original solution was 2.5 x 10¹¹particles/ml with a peak value of 128 nm,corresponding to the size of mesenchymal stem cell exosomes.（3）Protein immunoblotting showed positive expression of CD9 and CD63,consistent with specific positive markers on the surface of mesenchymal stem cell exosomes.Combining the above results suggests that the extracted vesicle-like substance conforms to the basic characteristics of ADSCs-exo.3.A rat model of CLP sepsis was successfully prepared.The 72-hour survival rates of the 3 groups of rats were 100%,40%,and 60%respectively.Compared with the Sham group,the survival time of CLP rats was significantly shortened,which is statistically significant（P<0.05）;Compared with the CLP group,the survival time of CLP rats was significantly lengthen,but were not statistically（P>0.05）.4.The expression level of inflammatory factors in serum of 48h after CLP surgery,ELISA test results:Compared with the Sham group,the levels of TNF-α,IL-1β,IL-6,and IL-10 in the CLP group and EXO group serum were all statistically significant（P<0.05）;compared with the CLP group,48h TNF-α,IL-1β,and IL-6 levels in EXO group serum decreased significantly,and IL-10 levels increased markedly,all of which were statistically significant（P<0.05）.5.Results of HE staining of small intestine,liver,and kidney tissue 48h after CLP surgery:Compared with the Sham group,the damage to the small intestine,liver,and kidney tissue in the CLP group and the EXO group was severe.Pathological damage scores all increased significantly,and there were statistical differences（P<0.05）;compared with the CLP group,the degree of damage to the small intestine,liver,and kidney tissue in the EXO group decreased,and there were statistical differences（P<0.05）.6.q PCR results of small intestinal tissue 48h after CLP surgery:Compared with the Sham group,the m RNA expression levels of TNF-α,IL-1β,IL-6,IL-4,and IL-10 in the CLP group and EXO group were all statistically significant.TNF-α,IL-1β,IL-6,IL-10 in the CLP group were statistically significant（P<0.05）,IL-4were not statistically（P>0.05）.TNF-α,IL-1β,IL-6 in the EXO group were not statistically significant（P>0.05）,IL-4,IL-10 are all statistically significant（P<0.05）;compared with the CLP group,the m RNA expression levels of TNF-α,IL-1β,and IL-6 in the EXO group decreased significantly,which is statistically significant（P<0.05）.The level of m RNA expression of IL-4 and IL-10 increased significantly,which is statistically significant（P<0.05）.7.Western blot results of small bowel tissue 48h after CLP surgery:Compared with the Sham group,the expression levels of TNF-α,IL-1β,IL-6,IL-4,and IL-10 proteins in the CLP group were significantly increased.The expression of IL-4 and IL-10proteins in the CLP group was not statistically significant（P>0.05）;compared with the CLP group,the expression levels of TNF-α,IL-1β,IL-6 proteins in the EXO group were significantly reduced,and IL-6 protein expression levels were significantly reduced,and IL-6 protein expression levels in the CLP group were significantly reduced.The levels were not statistically significant（P>0.05）;the rest were statistically significant（P<0.05）.The level of IL-4 and IL-10 protein expression increased significantly,which was statistically significant（P<0.05）.Conclusions:1.ADSCs were successfully cultivated in vitro using the tissue block affixing method,the expression of surface-specific markers was identified by flow cytometry,and the ability to form fat and osteogenesis determined the potential for multi-directional differentiation;2.ADSCs-Exo was extracted using differential centrifugation,and transmission electron microscopy,NTA,and Western Blot successfully identified ADSCS-exo;3.The caecal ligation perforation method successfully constructed a rat model of sepsis;4.ADSCs-exo can improve the functional damage of multiple organs such as intestines,liver,and kidneys in CLP sepsis rats,reduce mortality,lower the expression of pro-inflammatory factors（IL-1β,IL-6,TNF-α）and increase the expression of anti-inflammatory factors（IL-10,IL-4）,and reduce the inflammatory response.