| Laparoscopic surgical techniques can greatly reduce surgical injury,bleeding and postoperative adhesions,alleviate surgical pain and accelerate postoperative recovery,and therefore have been widely used in hepatobiliary surgery.However,it is still difficult to avoid ischemia reperfusion injury(IRI)during operation on liver,which affects postoperative liver tissue activity and liver function recovery and may even lead to liver failure.At present,studies on the mechanism of hepatic IRI and its prevention and control measures are still in the exploratory stage.Solution for how to reduce hepatic IRI and how to promote damage repair is one of the urgent problems to be solved in the field of medical science,veterinary medicine and comparative medicine.Mesenchymal stem cells(MSCs)have the potential of self-renewal and multi-directional differentiation,and can promote the repair and regeneration of injured tissues.Exosomes are nanoscale vesicles produced by cells through cytosis and cytosol,which are capable of carrying biological functions of parental cells and conducting cell-to-cell communication by loading proteins and nucleic acids.MSCs may mainly exert functions through paracrine mechanisms and are one of the important components of the paracrine fraction.MSCs derived exosomes(MSCs-exo)have protective effects in various organ injury such as brain,lung,kidney and liver,and can play anti-inflammatory,anti-oxidant,anti-apoptotic and regeneration roles.At present,MSCs-exo has become a research hotspot in the field of tissue regeneration and has a broad research prospect.Minipigs are ideal experimental animals in comparative medicine because of their closer homology to humans,as well as their similar body wight and anatomy structure to those of humans.Recently,researches on MSCs-exo mainly focus on rodents,and no report has been published on the application of MSCs-exo in liver IRI of middle or large animals.Adipose derived MSCs have become the seed cells in the field of regenerative medicine due to the wide source and easy accessibility of adipose tissue.Therefore,this experiment aimed to investigate and compare the intervention effects of ADSCs-exo and ADSCs on liver injury and their mechanisms of effects in minipigs based on a minipig model of partial hepatectomy combined with IRI by using laparoscopic techniques.The study selected 30 Bama minipigs as experimental animals,six of which were randomly taken to collect adipose tissue.ADSCs were isolated and extracted by collagenase digestion,and the specific surface antigens of MSCs were detected by flow cytometry.The lipogenic and hepatogenic differentiation ability of ADSCs was identified by induction medium and specific staining.ADSCs-exo was extracted from ADSCs culture supernatant by differential ultracentrifugation combined with 100 k Da ultrafiltration.The morphology of ADSCs-exo was characterized by electron microscopy,and the particle size of ADSCs-exo was identified using nanoparticle tracking analysis.The exosome-specific antigen was detected by Western Blot method.Another 24 minipigs were randomly divided into 4 groups,including the sham operation group(Sham group),the model group(IRI group),ADSCs-exo intervention group(Exo group),and ADSCs intervention group(ADSC group),with 6 pigs in each group.In Sham group,the surgical access and pneumoperitoneum were just established and the liver lobe was turned;In IRI group,the model of the laparoscopic left hepatectomy combined with the right hepatic IRI was established;The modeling method of Exo group and ADSC group was similar to that of IRI group.5 m L of PBS was injected into the Sham group and the IRI group.The Exo group was injected with 5 m L of PBS mixture containing 5×10~9 particles/kg ADSCs-exo,and the ADSC group was injected with 5 m L of PBS mixture containing 1×10~6 cells/kg ADSCs.Blood samples and liver tissue samples were collected before surgery,and on Day 1,Day 3 and Day 7 after surgery for blood routine,liver function test,morphological observation of liver histology,ROS content detection,TUNEL staining of apoptotic cells and Ki67 staining of regenerating cells,respectively,so as to evaluate the intervention effect of ADSCs-exo versus ADSCs.According to the above test results combined with the mechanism of liver IRI,the relevant indexes of oxidative stress,inflammation,mitochondrial fusion and division,mitochondrial biosynthesis,endoplasmic reticulum stress response,apoptosis,pyroptosis and liver regeneration were tested respectively to compare and analyze the regulatory effect of ADSCs-exo and ADSCs on liver injury.The results of this study are reported as follows:(1)Liver histology morphological observation results:Light microscopy findings showed disorganized hepatocyte cords,disappearance of hepatic sinusoids,severe hepatocyte swelling and vacuole-like changes,focal or punctate apoptosis or necrosis and inflammatory cell infiltration in the IRI group.Transmission electron microscopy observation revealed swollen hepatocytes,crinkled hepatocyte nuclei,significantly swollen mitochondria,and severely dilated endoplasmic reticulum in the IRI group.Compared with the IRI group,ADSCs-exo and ADSCs interventions could reduce the number of apoptotic or necrotic cells and alleviate the degree of inflammatory infiltration,and to decrease the degree of hepatocyte and mitochondrial swelling and endoplasmic reticulum expansion.(2)Liver function test results:The levels of ALT,AST,LDH,ALP,TBIL,and HA in serum were increased and the level of TP was decreased in each group after the liver injury.Compared with the IRI group,ADSCs-exo and ADSCs interventions could effectively reduce the levels of ALT,AST,LDH,ALP,TBIL,HA and maintain the level of TP.There was no significant difference between the two interventions in terms of their effect.(3)The results of oxidative stress level assay:The content of oxidation products ROS and MDA were increased and the activities of antioxidant enzymes SOD,CAT and GSH-PX were decreased in liver tissues of all groups after liver injury.Compared with the IRI group,the ADSCs-exo and ADSCs interventions were able to reduce the content of oxidation products and effectively improve the activity of antioxidant enzymes.(4)Results of inflammation level tests:The number of WBC,NE and MO in peripheral blood increased,while the number of LY decreased in all groups after liver injury.The levels of TNF-α,IL-1β,IL-18,IL-6,CRP and IL-10 in serum increased,and the expression levels of m RNA of inflammatory factors in liver tissues were consistent with the results in serum.Compared with the IRI group,ADSCs-exo and ADSCs interventions could effectively reduce the levels of pro-inflammatory factors,such as TNF-α,IL-1β,IL-18,IL-6 and CRP,and enhance the expression of anti-inflammatory factor IL-10,thereby effectively decreasing the inflammation levels in liver tissue and over the whole body.(5)The results of mitochondrial and endoplasmic reticulum injury repair-related indexes:The m RNA and protein levels of Opa1,Mfn1 and Mfn2,PGC1α,Nrf1 and Tfam in liver tissues of each group were decreased,while that of Drp1 and Fis1 were increased in liver tissues of all groups after liver injury,and ATP content was decreased.The m RNA and protein levels of GRP78,IRE1α/XBP1,PERK/e IF2α/ATF4,JNK,and CHOP were elevated.Compared with the IRI group,ADSCs-exo and ADSCs interventions were able to promote mitochondrial fusion and mitochondrial biosynthesis,inhibit mitochondrial division,and promote the recovery of mitochondrial function,and meanwhile reduce the endoplasmic reticulum stress response.(6)Results of apoptosis and pyroptosis-related indexes:The positive rate of TUNEL was increased in all groups after liver injury.The m RNA and protein levels or enzyme activities of Fas,Fasl,Bax,Cyt C,APAF1,P53,Caspase-3,Caspase-8,Caspase-9 were increased,but the m RNA and protein level of Bcl-2 was decreased.The m RNA levels and protein levels of TLR4,My D88,ASC,NLRP3,Caspase-1,GSDMD,and HMGB1 were elevated,and the m RNA level of NF-κB and the protein level of p-IκB were also raised.Compared with the IRI group,ADSCs-exo and ADSCs intervention inhibited the activation of pro-apoptotic factors and the activation of Caspase enzymes,and promoted the expression of anti-apoptotic factor Bcl-2.At the same time,the intervention could inhibit the activation of NLRP3 inflammasome and GSDMD,suppress the transcription and expression of inflammatory factors,and reduce cell mortality.(7)The results of liver regeneration-related indexes:The positive rate of Ki67 was increased in all groups after liver injury.The m RNA and protein levels of PCNA,Cyclin D1,HGF,STAT3,SOCS3,VEGF,ANG1,ANG2,and TGF-βwere increased.Compared with the IRI group,ADSCs-exo and ADSCs interventions were able to induce cells entering the cell cycle and trigger liver regeneration,while suppress the expression of the negative regenerative regulators SOCS3 and TGF-β,so as to promote the proliferation of hepatocytes and sinusoidal endothelial cells.There was no significant difference in the effect of ADSCs-exo versus ADSCs intervention on the regeneration level of the liver.In conclusion,ADSCs-exo and ADSCs interventions can improve liver injury of minipigs that underwent laparoscopic partial hepatectomy combined with IRI and can promote postoperative liver tissue morphology and liver function recovery effectively.ADSCs-exo intervention can effectively reduce the levels of oxidative stress,inflammation,apoptosis and pyroptosis after liver injury,regulate the expression of molecules related to mitochondrial and endoplasmic reticulum injury and recovery,thus promoting the level of liver regeneration,which is similar to the mechanisms and intervention effects of ADSCs,indicating that ADSCs-exo intervention may be an effective alternative to ADSCs in treating liver injury. |
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