| Partial hepatic resection is one of the common treatment methods for liver diseases,but due to the complex vascular structure and rich blood supply in the liver,partial hepatic resection is often accompanied by hepatic ischemia reperfusion injury(HIRI).Therefore,prevention and treatment of HIRI is an urgent problem to be solved in liver surgery.Mitochondrial autophagy has two sides.Moderate mitophagy can degrade aging or excess mitochondria in cells and recycle them to maintain the stability of the intracellular environment.However,excessive mitochondrial autophagy will aggravate cell damage and start the apoptosis process.Adipose mesenchymal stem cell-derived exosomes(ADSCs-exo)have the advantages of low immunogenicity,convenient storage,and no ethical restrictions.Studies have confirmed that ADSCs-exo can reduce HIRI by inhibiting oxidative stress,inflammatory response,apoptosis and other ways.Miniature pigs are widely used in comparative medicine due to their high homology and similar anatomical structure.However,the effect of ADSCsexo on mitophagy in pig HIRI injury has not been reported.Therefore,in this study,laparoscopic technology was used to establish a small porcine hepatic I/R combined with partial resection model and ADSCs-exo and ADSCs intervention were applied to explore the effect of ADSCs-exo on mitophagy in this model.The intervention effects of ADSCS-exo and ADSCs were further compared.Twenty-four healthy Guangxi Bama mini-pigs(20-30 kg)were divided into Sham group(Sham group),model group(IRI group),ADSCS-exo intervention group(EXO group)and ADSCs intervention group(ADSCs group).Dissolve ADSCs exo and ADSCs in 5ml 37 ℃sterile PBS at a certain dose.In IRI group,EXO group and ADSCs group,the left liver was resected after 60 minutes of right liver ischemia,and sterile PBS,ADSCs-exo(5×109 /kg)and ADSCs(1×106 /kg)were injected into the portal vein after the left liver was resected.Sham group only created the surgical approach,flipped the liver lobe,and maintained pneumoperitoneum and anesthesia for the same time.Serum and liver tissue samples were collected preoperatively,1 d,3 d and 7 d postoperatively.The kit assays peroxidase(MPO)and catalase(CAT)activities.RT-q PCR,Western Blot and immunofluorescence methods were used to detect the gene and protein levels of mitophagy related factors Beclin1,ATG5,ATG12,LC3,p62,PINK1,Parkin,BNIP3,FUNDC1 and PI3K-AKT-m TOR signaling pathway.The test results are as follows:(1)Electron microscope results: The structure of liver cells was normal before operation and on the 1 day,3 days and 7 days after operation in Sham group.No mitochondrial autophagy was found.On the 1 day after operation,mitochondria in IRI group were swollen,mitochondria disappeared,mitochondrial membrane was blurred and mitochondrial autophagy structure could be observed.Mitochondrial crest in EXO group and ADSCs group decreased but changed slightly compared with IRI group and there was no mitochondrial autophagy structure.On the3 days after operation,the mitochondrial swelling in IRI group still showed the mitochondrial autophagy structure,while the mitochondrial swelling in EXO group and ADSCs group was lighter than that in IRI group,and there was no mitochondrial autophagy structure.7 days after operation,the structure of hepatocytes in IRI group,EXO group and ADSCs group was basically normal and there was no mitochondrial autophagy structure.(2)Oxidative stress results: 1 day after surgery,the serum MPO activity in IRI group was significantly increased compared with that in Sham group;Compared with IRI group,EXO group and ADSCs group significantly decreased,and EXO group and ADSCs group had no significant difference.There was no significant difference in serum MPO activity at other time points.On 1 day after surgery,compared with Sham group,serum CAT activity of IRI group,EXO group and ADSCs group was significantly decreased.Compared with IRI group,serum CAT activity of EXO group and ADSCs group was significantly increased.There was no significant difference between EXO group and ADSCs group.3 days after surgery,serum CAT activity in IRI group was significantly lower than that in Sham group.Compared with IRI group,serum CAT activity of EXO group and ADSCs group was significantly increased.There was no significant difference between EXO group and ADSCs group.There were no significant differences in serum MPO and CAT activities at other time points.(3)Mitophagy related results: On1 day and 3 days after surgery,compared with the Sham group,the expression levels of mitophagy related genes Beclin1,ATG5,ATG12,LC3,PINK1,Parkin,BNIP3 and FUNDC1 in IRI group,EXO group and ADSCs group were significantly increased,while the gene expression level of p62 was significantly decreased.Compared with IRI group,the m RNA expression levels of Beclin1,ATG5,ATG12,LC3,PINK1,Parkin,BNIP3 and FUNDC1 in EXO group and ADSCs group were significantly decreased,while the m RNA expression levels of p62 were significantly increased.On1 day and 3 days after surgery,compared with the Sham group,the expression levels of mitophagy related proteins Beclin1,PINK1,Parkin and BNIP3 and LC3Ⅱ/Ⅰ ratio of IRI group,EXO group and ADSCs group were significantly increased,while the protein expression level of p62 was significantly decreased.Compared with IRI group,the protein expression levels of Beclin1,LC3,PINK1,Parkin and BNIP3 in EXO group and ADSCs group were significantly decreased,and the LC3Ⅱ/Ⅰ ratio was also significantly decreased,while the protein expression level of p62 was significantly increased.On day 1 after surgery,compared with Sham group,the fluorescence density ratio of PINK1/VDAC1 in IRI group,EXO group and ADSCs group was significantly increased;Compared with IRI group,EXO group and ADSCs group were significantly lower.Three days after surgery,compared with Sham group,the fluorescence density ratio of PINK1/VDAC1 in IRI group was significantly increased,while there was no significant difference between EXO group and ADSCs group.Compared with IRI group,EXO group and ADSCs group were significantly lower.Compared with ADSCs group,the expression levels of the above genes and proteins in EXO group had no significant difference at 1 day and 3 days.after surgery There were no significant differences in the expression levels of these genes and proteins among groups before operation and at 7 days after surgery.(4)PI3K-AKT-m TOR detection results: Compared with the Sham group,the expression levels of PI3 K,AKT and m TOR genes in IRI group,EXO group and ADSCs group were significantly decreased on day 1 and 3 after surgery.Compared with IRI group,the expressions of PI3 K,AKT and m TOR genes in EXO group and ADSCs group were significantly increased.On day 1 and 3 after surgery,compared with Sham group,the relative expression of p-PI3K/PI3 K,p-Akt /AKT and p-m TOR/m TOR proteins in IRI group,EXO group and ADSCs group was significantly decreased.Compared with IRI group,the relative expressions of p-PI3K/PI3 K,p-Akt /AKT and p-m TOR /m TOR proteins in EXO group and ADSCs group were significantly increased.The expression levels of genes and proteins in EXO group and ADSCs group were not significantly different.There were no significant differences in the expression levels of genes and proteins in the pathway before and 7 d after surgery.In conclusion: In the model of small pig liver I/R combined with hepatectomy,portal vein injection of ADSCS-exo and ADSCs can reduce the autophagy structure of liver cells,inhibit oxidative stress,inhibit mitochondrial autophagy,and activate the PI3K-AKT-m TOR signaling pathway to protect the liver.These results indicate that ADSCS-exo and ADSCs may have antioxidant effects and inhibit excessive mitophagy through PI3K-AKT-m TOR signaling pathway,and there is no significant difference in the intervention effect between the two.Therefore,ADSCS-exo can achieve the expected effect of alleviating the damage of excessive mitophagy in hepatic I/R combined with partial hepatectomy. |
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