Investigation Of The Protective Mechanism Of Drug-Loaded ADSCs-Exo Based On AMPK/SIRT1/PGC-1α Pathway Against Hepatic IRI Combined With Partial Resection Injury In Rats

Hepatic resection/transplantation,as an effective approach to remove liver tumors and treat liver failure,cirrhosis and hepatocellular carcinoma,inevitably leads to ischemia reperfusion injury(IRI)in the remaining liver tissue/donor liver.Since hepatic IRI severely leads to postoperative liver dysfunction and there is a lack of effective therapeutic drugs due to the complex mechanism of IRI,targeting to mitigate the damage caused by hepatic IRI and promoting the repair of postoperative liver injury is a hot content in recent years and has important clinical significance.Adipose mesenchymal stem cell derived exosomes(ADSCs-Exo)and metformin(Met)can play a mitochondrial protective role in the treatment of hepatic IRI,but whether there is a synergistic mechanism for their intervention is not yet known.Combining the unique advantages of exosomes as drug carriers,the aim of this study was to investigate the protective effects and mechanisms of the constructed Met and ADSCs-Exo complex(Met-Exo)on the liver IRI combined with partial resection injury in rat and hypoxic reoxygenation injury of rat primary hepatocytes(HCs).The main experiments were as follows:(1)In vivo experiment,30 male SD rats were randomly divided into 5 groups: Sham group,Model group,Met group(50 mg/kg),ADSCs-Exo group(100 μg/each),and Met-Exo group(100μg/each).Rats in the Sham group were only anesthetized,and the liver lobes were opened and turned over.Rats in the Model,Met,ADSCs-Exo and Met-Exo groups were established as a model of combined hepatic left lobe resection injury with 30 min ischemia reperfusion in the middle and left lobes of the liver.Each group received a 1 mL solution of PBS containing the intervention materials via tail vein injection in the immediate postoperative period.Blood and liver samples were taken at 24 h of reperfusion to observe the changes in microstructure and ultrastructure of liver tissue,detect the changes in biochemical indexes of liver function and blood routine indexes,detect the changes of oxidative stress and inflammation-related factors,and detect the expression of mitochondrial function,mitochondrial biosynthesis and dynamics,autophagy and related factors in AMPK/SIRT1/PGC-1α pathway in liver tissue.(2)To further verify whether ADSCs-Exo and Met play a synergistic role in the protection of hepatic IRI through AMPK/SIRT1/PGC-1α pathway.In vitro experiments were performed in rat HCs,divided into six groups: Con group,H/R group,Met group(1 mM),ADSCs-Exo group(10 μg/mL),Met-Exo group(10 μg/ mL)and EX-527 group(10 mM EX-527+10 μg/mL Met-Exo).Cell viability,toxicity,hepatocyte function,oxidative stress and inflammatory factors,mitochondrial function,biosynthesis and dynamics,cellular autophagy and AMPK/SIRT1/PGC-1α pathway related factors were measured in each group by taking culture supernatant and cell samples respectively.The test results were as follows:(1)The serum levels of ALT,AST,TBIL,ALP,and LDH were highly significantly elevated in the Model group,and Met,ADSCs-Exo,Met-Exo interventions reduced the levels of the above indices,and Met-Exo had a stronger ability to promote liver function recovery.The protective effect of Met-Exo was inhibited by EX-527 treatment in the in vitro test.It indicates that Met-Exo may have a protective effect on hepatocytes by regulating AMPK/SIRT1.(2)In the Model group,hepatocytes showed watery degeneration,swelling,disorganized arrangement,nuclear lysis,localized necrotic foci and inflammatory cell infiltration,while in the Met,ADSCs-Exo and Met-Exo intervention groups,the degree of liver damage was significantly reduced,as evidenced by mild swelling of hepatocytes,reduced watery degeneration and reduced inflammatory cell infiltration.(3)The changes in SOD and CAT activities,T-GSH/GSSH ratio,GSH content decreased,MPO activity,MDA and ROS content increased in the liver tissues of rats in the Model group.The changes in the above indexes were reversed after Met,ADSCs-Exo and Met-Exo interventions,in which Met-Exo showed significant advantages in increasing SOD activity and decreasing MPO activity.Both Met and Met-Exo interventions enhanced antioxidant enzyme activities and reduced oxidative stress product production,and EX-527 treatment led to a decrease in the antioxidant capacity of Met-Exo in vitro assays.It indicates that Met-Exo may inhibit oxidative stress response by regulating AMPK/SIRT1.(4)The number of WBC and NE in the blood of Model group rats was highly significantly increased,the number of LY was significantly decreased,and the levels of pro-inflammatory factors TNF-α,IL-1β and IL-6 in liver tissue were highly significantly increased,and the levels of antiinflammatory factor IL-10 were highly significantly decreased.Met,ADSCs-Exo and Met-Exo interventions could reduce inflammatory factors,and increase the levels of anti-inflammatory factors,with Met-Exo showing a significant advantage in reducing IL-1β,IL-6,and increasing IL-10 levels.In an in vitro test,EX-527 antagonized Met-Exo.This suggests that Met-Exo may reduce the inflammatory response by regulating AMPK/SIRT1.(5)In the Model group,ATP content was significantly reduced in liver tissues,and mitochondria were swollen,cristae were broken,and bilayer structures were dissolved.Met,ADSCs-Exo,and Met-Exo interventions increased ATP content and were statistically significant,and mitochondrial morphology was significantly improved.The effect of Met-Exo intervention was suppressed by EX-527 treatment in the in vitro assay.This suggests that Met-Exo may protect mitochondrial structure and function by regulating AMPK/SIRT1.(6)Ischemia/hypoxia treatment resulted in decreased Nrf1,Tfam protein expression,and Met,ADSCs-Exo,and Met-Exo interventions increased Nrf1,Tfam protein expression.Among them,Met-Exo showed a significant advantage in increasing Nrf1,Tfam protein expression.The effect of Met-Exo intervention was suppressed by EX-527 treatment in the in vitro assay.It indicates that Met-Exo may promote mitochondrial biosynthesis by regulating AMPK/SIRT1.(7)Ischemia/hypoxia treatment resulted in a highly significant increase in Fis-1 protein expression and a highly significant decrease in p-Drp1 ser637,Opa1,and Mfn2 protein expression,and Met,ADSCs-Exo,and Met-Exo interventions reversed the changes in the above indices,with Met-Exo having a significant advantage over Met and ADSCs-Exo,and EX-527 treatment caused the effect of Met-Exo intervention to be suppressed.It indicates that Met-Exo may promote mitochondrial fusion and inhibit mitochondrial division by regulating AMPK/SIRT1.(8)Ischemia/hypoxia treatment resulted in a highly significant decrease in LC3B-II and Beclin-1 protein expression and a highly significant increase in p62,p-m TOR,and p-S6K1/S6K1 protein expression.Met-Exo intervention reversed these index changes and had a stronger effect than Met and ADSCs-Exo intervention,and EX-527 treatment caused the effect of Met-Exo intervention to be suppressed.This suggests that Met-Exo may promote cellular autophagy by regulating AMPK/SIRT1.(9)Ischemia/hypoxia treatment resulted in a highly significant decrease in p-AMPK/AMPK,SIRT1,and PGC-1α protein expression.p-AMPK/AMPK,SIRT1,and PGC-1α protein expression were promoted by Met-Exo intervention,which had a significant advantage over Met and ADSCsExo intervention,and the effect of Met-Exo intervention was made by EX-527 treatment was suppressed.It was demonstrated that Met and ADSCs-Exo exerted synergistic promotion effects in activating AMPK,SIRT1,and PGC-1α,and Met-Exo could effectively regulate AMPK/SIRT1/PGC-1α signaling pathway.In summary,Met-Exo intervention can protect against hepatic IRI combined with partial resection injury in rats and is more effective than Met and ADSCs-Exo,by activating the AMPK/SIRT1/PGC-1α pathway to maintain mitochondrial function,promote mitochondrial biosynthesis and fusion,inhibit mitochondrial division,promote autophagy,reduce tissue environment inflammation and oxidative stress,ameliorate tissue microstructural and ultrastructure damage,and it promotes the recovery of hepatocyte and liver tissue function.This indicates that ADSCs-Exo can not only play a targeting role as a drug carrier but also has a great potential to act as a vehicle to act synergistically with drugs in the treatment of tissue and organ damage,which provides a new therapeutic strategy and experimental basis for the treatment of liver injury in medical science and clinical veterinary.