Protective Effect Of Exosomes From Adipose Derived Stem Cells On Hepatic Ischemia Reperfusion Combined With Partial Hepatectomy Injury In Rats

Hepatic ischemia reperfusion(I/R)injury is an inevitable pathophysiological process during liver resection and liver transplantation,and it is a major factor leading to postoperative liver dysfunction.At present,the pathogenesis of hepatic I/R injury has not been fully understood,and effective treatment measures and drugs are lacking.How to alleviate hepatic I/R injury is an urgent problem to be solved in the veterinary medicine and even medical research.Adipose derived mesenchymal stem cells(ADSCs)have the potential of self-renewal and multidirectional differentiation and the properties of promoting the repair and regeneration of injured tissues.Studies have shown that ADSCs exert biological functions through paracrine action,and exosomes are the main components of paracrine substances.In recent years,it has been found that ADSCs derived exosomes(ADSCs-exo)has the function of promoting the repair of damaged tissues in the nervous system,myocardial injury,lung injury,and kidney injury.However,there are few studies on the protective effect of ADSCs-exo on hepatic injury.Therefore,this study aims to explore the protective effect and related mechanism of ADSCs-exo on hepatic I/R combined with partial hepatectomy injury in rats.The main research contents were as follows:(1)To establish an ideal model of hepatic I/R combined with partial hepatectomy,72 male SD rats were randomly divided into three groups:Sham group,I60R+PH group,and I30R+PH group.In the Sham group,only the first hepatic hilum was exposed without blocking blood supply.In the I60R+PH group and I30R+PH group,middle lobe and left lobe of the liver were respectively ischemic for 60 min and 30 min,and the left lobe of liver was resected.By observing the changes of liver histopathology,liver function,blood routine,oxidative stress,inflammatory response,Caspase activity and ATP content at 6 h,24 h,72 h and 168 h after reperfusion,the best model group was selected for the next test.(2)To explore the protective effect and mechanism of ADSCs and ADSCs-exo on hepatic injury,24 male SD rats were randomly divided into four groups:Sham group,I30R+PH group,ADSCs group,and ADSCs-exo group.In the Sham group,only the first hepatic hilum was exposed without blocking blood supply and was injected with 600μl PBS through the tail vein.In the I30R+PH group,ADSCs group,and ADSCs-exo group,middle lobe and left lobe of the liver were ischemic for 30 min,and the left lobe of liver was resected.The rats in the three groups were injected respectively with 600μl PBS,2×10~6ADSCs in 600μl PBS,and 100μg ADSCs-exo in 600μl PBS through the tail vein.The effect of ADSCs and ADSCs-exo on liver injury in rats was analyzed comparatively by observing the changes of liver microstructure and ultrastructure,the changes of factors related to oxidative stress and inflammatory response,and the expression of related indicators of apoptosis,mitochondrial function,mitochondrial biogenesis,mitochondrial dynamics and endoplasmic reticulum stress at 24 h after reperfusion.The results were as follows:(1)Effect of ischemic time on hepatic I/R combined with partial hepatectomy injury in ratsCompared with the I30R+PH group,ALT level increased significantly at 6 h and 24 h after reperfusion in the I60R+PH group,furthermore,Caspase-9 activity increased significantly at 6 h after reperfusion and Caspase-3 activity increased significantly at 6 h and 24 h after reperfusion,however,SOD and GSH-px activities decreased significantly at 6 h after reperfusion.The levels of inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-10 increased,and ATP content decreased in the I60R+PH group when compared with the I30R+PH group.Hepatocyte necrosis was observed at 6and 24 h after reperfusion,the degree of which in the I60R+PH group was greater than that in the I30R+PH group.These results showed that hepatic I/R combined with partial hepatectomy injury in rats resulted in more severe injury within 24 h,and indicated that hepatic injury was aggravated with longer ischemic time.Therefore,the I30R+PH group is the best experimental model after optimization test.(2)Protective effect of ADSCs-exo on hepatic I/R combined with partial hepatectomy injury1)The levels of ALT,AST,TBIL,ALP and LDH increased significantly in the I30R+PH group.After treatment with ADSCs and ADSCs-exo,the levels of ALT,AST,TBIL,ALP and LDH were reduced significantly,promoting the recovery of liver function.2)In the I30R+PH group,there were multiple necrotic foci,inflammatory cell infiltration,and severe swelling of hepatocytes.Transmission electron microscopy revealed that nuclear shrinkage,chromatin marginalized aggregation,obviously swelling of mitochondria and severely expansion of endoplasmic reticulum in the I30R+PH group.Treatment with ADSCs and ADSCs-exo improved microstructural and ultrastructural damage,which was manifested as a small amount of inflammatory cell infiltration,mild swelling of hepatocytes,slight nuclear shrinkage,improved mitochondrial damage,and ameliorated endoplasmic reticulum expansion.3)The activities of antioxidant enzymes SOD,CAT and GSH-px of the liver tissue in the I30R+PH group decreased significantly,and the contents of MDA and ROS increased significantly.After ADSCs and ADSCs-exo treatment,the activities of antioxidant enzymes increased significantly,and the contents of MDA and ROS in liver tissues decreased significantly.4)The number of WBC,NE and LY in blood increased significantly in the I30R+PH group.The contents of inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-10 in liver tissue increased significantly in the I30R+PH group.After treatment with ADSCs and ADSCs-exo,the number of WBC,and the content of pro-inflammatory factors in liver tissue decreased significantly,and the content of anti-inflammatory factors increased significantly.5)In the I30R+PH group,the number of apoptotic hepatocytes increased significantly,moreover,the m RNA and protein expression of Bax,Caspase-9,and Caspase-3 increased significantly,while Bcl-2 decreased significantly.ADSCs and ADSCs-exo significantly decreased the number of apoptotic hepatocytes and the expression of pro-apoptotic factors,and increased the expression of anti-apoptotic factors.6)In the I30R+PH group,ATP content in liver tissues decreased significantly,while increased significantly after ADSCs and ADSCs-exo treatment.7)In the I30R+PH group,the m RNA and protein expression of PGC-1α,Nrf1 and Tfam decreased significantly.After ADSCs and ADSCs-exo treatment,the m RNA and protein expression of PGC-1α,Nrf1 and Tfam increased significantly.8)In the I30R+PH group,Drp1 and Fis1 m RNA and protein expression increased significantly,whereas Mfn1,Mfn2 and Opa1 m RNA and protein expression decreased significantly.ADSCs and ADSCs-exo treatment significantly decreased the m RNA and protein expression of Drp1 and Fis1,and significantly increased Mfn1 and Opa1 m RNA expression and Mfn1,Mfn2 and Opa1 protein expression.9)GRP78,p-PERK/PERK,ATF-6,p-IRE1α/IRE1α,p-e IF2α/e IF2α,ATF-4,CHOP,XBP1s,p-JNK/JNK,Cleaved Caspase-12 protein expression in the I30R+PH group were significantly increased.GRP78,ATF-6,IRE1α,ATF-4,CHOP,XBP1,JNK and Caspase-12 m RNA expression were significantly increased in the I30R+PH group.ADSCs and ADSCS-Exo significantly inhibited the above m RNA and protein expression.In conclusion,ADSCs-exo injection through tail vein can inhibit oxidative stress and inflammatory response,improve microstructure and ultrastructure in liver tissue,and promote the recovery of liver function.ADSCs-exo can regulate mitochondrial biogenesis,promote mitochondrial fusion,inhibit mitochondrial fission,improve mitochondrial dysfunction,and inhibit endoplasmic reticulum stress and apoptosis,and play a protective role against hepatic I/R combined with partial hepatectomy injury,which indicated that promoting mitochondrial biogenesis,maintaining mitochondrial dynamics balance and alleviating endoplasmic reticulum stress may provide a novel therapeutic strategy for the prevention and treatment of hepatic I/R combined with partial hepatectomy injury.There was no significant difference in the intervention efficacy between ADSCs-exo and ADSCs,suggesting that ADSCs-exo may be a safe and effective alternative to ADSCs for treating hepatic I/R combined with partial hepatectomy injury.