| Liver ischemia reperfusion injury(LIRI)is one of the main causes of liver function injury and failure after primary liver transplantation and hepatectomy.Autophagy is a process associated with cell death and survival,which maintains the relative stability of intracellular energy by degrading intracellular compounds,clearing damaged organelles and protein aggregates.Severe ischemia can lead to over-enhancement of autophagy,which can destroy normal proteins and organelles in cells,leading to cell death.Nowadays,adipose-derived mesenchymal stem cells(ADSCs)have good therapeutic potential in various diseases and are considered as a novel drug for regenerative medicine.However,there is a hypothesis that the therapeutic effect of ADSCs may be mainly derived from their paracrine effects.Many studies have also demonstrated that adipose-derived mesenchymal stem cell-conditioned medium(ADSC-CM)contains many cytokines and chemokines.It has been found that ADSCs have a good therapeutic effect on hepatocyte autophagy injury caused by hepatic IRI combined with hepatectomy injury.Here,we propose a hypothesis that ADSC-CM has therapeutic effects on excessive hepatocyte autophagy injury caused by hepatic ischemia reperfusion injury(IRI)combined with hepatectomy injury.ADSCs may protect the liver and promote the process of liver tissue repair by treating excessive hepatocyte autophagy injury caused by hepatic IRI combined with hepatectomy injury through their paracrine effect.In addition,the therapeutic mechanism of ADSC-CM on excessive autophagy injury in hepatic IRI combined with hepatectomy was further explored.Twenty-four healthy minipigs were randomly divided into four groups(n=6): model group(IRI group),DMEM control group(DMEM group),ADSCs treatment group(ADSCs group)and ADSCsCM treatment group(CM group).Laparoscopic surgery was used to establish the same model in each group(right hemi-hepatic ischemia for 60 min combined with left hemi-hepatectomy),and the same dose of corresponding interventions was injected into the liver parenchyma immediately after surgery.The IRI group was injected with normal saline,the DMEM group was injected with DMEM,the ADSCs group was injected with ADSCs resuspension,and the CM group was injected with ADSC-CM concentrate.Liver tissue samples were taken from each group by laparoscopic surgery at preoperation,1 day after operation,3 days after operation and 7 days after operation.The ultrastructure of hepatocytes was observed by electron microscopy,and the changes of autophagy parameters in liver tissue samples were detected by RT-q PCR,Western blot and immunohistochemistry.Specific examination indexes included: autophagy related factors Beclin1,LC3 II,p62,ATG5,ATG12,autophagy pathway PI3K-AKT-m TOR related factors PI3 K,AKT,m TOR.The model was successfully established in this experiment,and the recovery was good after operation.The results of electron microscopy showed that obvious autophagic structure appeared in each group at 1 d after operation.The overall structure of hepatocytes in IRI group and DMEM group was severely damaged,mitochondria and endoplasmic reticulum swelled,and even mitochondrial cristae disappeared,cell nucleus collapsed and deformed at 1 d.The structure of hepatocytes in ADSCs group and CM group was relatively clear,mitochondria and endoplasmic reticulum slightly swelled,and the nucleus structure was basically normal.RT-q PCR results showed that there was no significant difference in the expression levels of autophagy-related genes(Beclin1,ATG5,ATG12,p62)and autophagy pathway-related genes(PI3K,AKT,m TOR)between DMEM group and IRI group at 1 d,3 d and 7 d after operation(p>0.05).At 1 d and 3 d,the expression levels of Beclin1,ATG5,ATG12 m RNA were significantly decreased(p<0.05),p62 m RNA was significantly increased(p<0.05),and the expression levels of PI3 K,AKT,m TOR m RNA were extremely significantly increased(p<0.01)in ADSCs group compared with IRI group;compared with DMEM group,the expression levels of Beclin1,ATG5,ATG12 m RNA were significantly decreased(p<0.05),p62 m RNA was significantly increased(p<0.05),and the expression levels of PI3 K,AKT and m TOR were significantly increased(p<0.01).At 7 d,there was no significant difference between the groups(p>0.05).There were no significant differences in the expression of autophagy-related genes Beclin1,ATG5,ATG12 and p62 between ADSCs group and CM group at 1 d,3 d and 7 d(p>0.05).Western-Blot results showed that there were no significant differences between DMEM group and IRI group in their respective autophagy-related proteins(Beclin1,LC3 II,p62),ratio of autophagy pathway-related proteins(p-AKT/AKT,p-m TOR/m TOR)at 1 d,3 d and 7 d(p>0.05).At 1 d and 3 d,the protein levels of Beclin1 and LC3 II were extremely significantly decreased(p<0.01),the protein levels of p62 were extremely significantly increased(p<0.01),and the ratios of p-AKT/AKT and p-m TOR/m TOR were extremely significantly increased(p<0.01)in the ADSCs group compared with the IRI group;compared with the DMEM group,the protein levels of Beclin1 and LC3 II were extremely significantly decreased(p<0.01),and the protein levels of p62 were extremely significantly increased(p<0.01),and the ratio of p-AKT/AKT and p-m TOR/m TOR was extremely significantly increased(p<0.01).At 7 d,there was no significant difference between the groups(p>0.05).There was no significant difference in the expression of autophagy-related proteins Beclin1,LC3 II and p62 at 1 d,3 d and 7 d(p>0.05).Immunohistochemical results showed that the results of LC3 II protein detection were consistent with their Western-Blot results.In summary:(1)ADSC-CM can significantly improve the excessive autophagy injury of hepatocytes in hepatic IRI combined with partial hepatectomy,and may inhibit the excessive autophagy injury of hepatocytes through the autophagy-related pathway PI3K-AKT-m TOR.(2)There was no significant difference in autophagy between ADSCs group and CM group.Therefore,it is speculated that ADSCs may improve the excessive autophagy injury of hepatocytes in hepatic IRI combined with partial hepatectomy through paracrine effect. |
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