Protective Effect Of Adipose-derived Stem Cell Condition Medium On Laparoscopic Hepatic Ischemia-reperfusion Injury Combined With Hepatectomy Of Miniature Pigs

The liver is the largest parenchymal organ in mammals.It has important functions of processing and storing nutrients,metabolizing drug poisons,secreting bile,and synthesizing proteins.Hepatic ischemia-reperfusion injury is a common clinical syndrome in liver surgery.Laparoscopic hepatectomy appeared in the 1990s.Laparoscopic surgery can minimize abdominal nerve and muscle damage and reduce bleeding,postoperative pain and adhesions,which can speed up recovery.Even with minimally invasive surgery,liver ischemia-reperfusion injury is still inevitable in clinical operations such as liver resection,liver transplantation,and hemorrhagic shock,which can seriously lead to hepatitis and liver failure after surgery.An effective treatment for advanced liver dysfunction is orthotopic liver transplantation.However,it faces the problems of organ shortage,high cost,immune rejection and transplant complications.Therefore,to find a safe and effective treatment that can reduce liver ischemia-reperfusion injury and improve liver regeneration ability is one of the problems to be solved in liver surgery.Adipose-derived mesenchymal stem cells(ADSCs)are capable of self-renewal and multi-directional differentiation.ADSCs are ideal seed cells based on cell therapy because they have rich sources of adipose tissue and have less damage to the body from obtaining adipose tissue.ADSCs play a role mainly including differentiation mechanisms and paracrine mechanisms.Paracrine effect refers to the biological effects of various biological factors released by stem cells on target cells in the microenvironment through the cell matrix in the local and adjacent tissues.With the study of paracrine mechanisms,it has been discovered that a variety of cytokines secreted by ADSCs can promote angiogenesis,inhibit inflammatory responses,regulate immune responses and other biological functions,and play an important role in repairing and regenerating damaged tissues.During the in vitro culture of adipose-derived mesenchymal stem cells,biologically active substances such as cytokines are secreted into the culture medium.This culture medium containing stem cells secretome is called adipose-derived mesenchymal stem cell condition medium(ADSC-CM).Although ADSCs have clinical effects on many diseases,ADSCs are still immunogenic,and uncontrolled differentiation of implanted ADSCs is still an important safety hazard.Therefore,cell-free therapy using ADSC-CM has become a regenerative medicine treatment method that avoids the limitations of cell therapy.At present,the animal models of liver ischemia-reperfusion injury are mainly rodents.Miniature pigs have good homology with humans,and their size and organ size are closer to humans.Therefore,it is an ideal experimental animal for studying ischemia-reperfusion injury.The experimental data obtained from the miniature pigs research is valuable and easy to be clinically transformed.ADSC-CM is less studied in large experimental animals,especially in the liver field.Therefore,the purpose of this study was to investigate the efficacy of ADSC-CM in treating hepatic ischemia-reperfusion combined with partial hepatectomy by establishing a model of hepatic ischemia-reperfusion combined with partial hepatectomy.It can provide research basis for comparative medicine to develop liver disease treatment and broaden ADSC-CM transplantation field.This study used 30 miniature pigs,6 of which were used for adipose tissue acquisition.ADSCs were isolated and cultured by collagenase digestion.Induction medium and specific staining were used to identify adipogenic,osteogenic,and hepatic differentiation abilities of ADSCs.Cell flow cytometry was used to identify ADSCs surface specific antigens.ADSCs were cultured by starvation for 48 h to obtain ADSC-CM.The concentrated ADSC-CM was obtained by centrifugation through3 kda ultrafiltration tubes for subsequent experiments.The other 24 miniature pigs were randomly divided into four groups:model group(IRI),DMEM control group(DMEM),adipose-derived mesenchymal stem cell group(ADSCs),and adipose-derived mesenchymal stem cell condition medium group(CM),6 animals per group.All experimental animals established a model of hepatic ischemia-reperfusion combined with partial hepatectomy by laparoscopy.IRI group was injected with normal saline through liver parenchyma.DMEM group was injected with basal medium.ADSCs group was injected with adipose mesenchymal stem cells at a dose of 1×10~6/kg.In the ADSC-CM group,the same amount of condition medium secreted by stem cells was extracted at a dose of 1×10~6/kg,and concentrated ADSC-CM was injected into the liver parenchyma.Blood samples and liver tissue samples were collected preoperation,postoperative 1 day,3 days and 7 days for the detection of related indicators.Histopathological changes were observed by HE staining.Ultrastructural changes were observed by transmission electron microscope.Inflammatory cell changes in peripheral blood were detected by a hemocytometer.Antioxidant enzyme activities and lipid peroxidation level were detected by commercial kits.TUNEL staining was used to detect apoptotic cell rates.Detection of changes in serum inflammatory factors,regenerative factors and ADSC-CM components by ELISA.Detection of inflammation,apoptosis and regeneration-related genes by q RT-PCR.Western Blot was used to detect apoptosis and regeneration-related protein levels in liver tissue,and immunohistochemical staining was used to detect apoptosis and regeneration-related proteins.The results as follows:(1)Histopathology and ultrastructural changes:hepatic lobular structures were damaged,hepatocytes were arranged disorderly,hepatocyte vacuole degeneration was obvious,and infiltration of inflammatory cells appeared postoperative.Compared with IRI group and DMEM group,ADSCs or ADSC-CM intervention significantly reduced vacuolar degeneration and infiltration of inflammatory cells.After operation,the endoplasmic reticulum of hepatocytes in IRI group and DMEM group was severely expanded,the nuclear membrane was shrunk,and chromatin was concentrated to the edge.After intervention with ADSCs or ADSC-CM,the endoplasmic reticulum was slightly dilated and the nuclear membrane shrinkage and chromatin aggregation of hepatocytes were improved.(2)Liver function:ALT,AST,LDH,ALP,and T-BIL levels increased in all groups after surgery,but TP levels decreased.Compared with IRI group and DMEM group,ADSCs or ADSC-CM intervention improve liver function after surgery that significantly reduced ALT,AST,LDH,ALP,T-BIL levels and alleviate the decrease in TP level,promote the restoration of liver function.(3)Changes in oxidative stress indicators:Antioxidant enzymes CAT,GSH-Px and SOD enzyme activity in liver tissues of all groups were significantly reduced,and MPO enzyme activity and MDA content were significantly increased after surgery.After intervention with ADSCs or ADSC-CM,the antioxidant enzyme activity was significantly enhanced,the MPO enzyme activity was reduced,and the level of the oxidation product MDA was reduced compared with IRI group and DMEM group.Moreover,there was no significant difference in the above indicators of oxidative stress between the two intervention groups.(4)Changes in inflammatory indicators:The levels of inflammatory cells WBC,NE,and LY in peripheral blood increased rapidly after operation.After intervention with ADSCs or ADSC-CM,the number of inflammatory cells in peripheral blood was reduced.The levels of inflammatory factors TNF-?,IL-1?,IL-6 and IL-10 increased in the serum of IRI group and DMEM group.After intervention with ADSCs or ADSC-CM,the levels of the pro-inflammatory factors,such as TNF-?,IL-1?,and IL-6 were reduced,while the levels of the anti-inflammatory factor IL-10 were increased.The expression of inflammatory factor genes in liver tissue was consistent with the serum results.ADSCs and ADSC-CM significantly improved the postoperative inflammation response by inhibiting the excessive release of pro-inflammatory factors and enhancing the expression of anti-inflammatory factors.Moreover,there was no significant difference between the two intervention groups.(5)Changes in apoptosis indicators:The levels of P53,Bax,Fas,Fasl genes and proteins in liver tissues of all groups were significantly increased,and Bcl-2 expression level was decreased.Compared with IRI group and DMEM group,ADSCs or ADSC-CM intervention inhibited the expression of apoptosis-related indicators P53,Bax,Fas,Fasl,and increased the expression of anti-apoptotic protein Bcl-2;ADSCs and CM groups also significantly reduced the rate of TUNEL-positive cells and caspase enzyme activity,reduce apoptosis of hepatocytes after surgery.Moreover,there was no significant difference between the two intervention groups.(6)Changes in regeneration indicators:Compared with the IRI group and the DMEM group,ADSCs or ADSC-CM intervention significantly increased the expression levels of angiogenesis-related factors VEGF,ANG-1,and ANG-2 in serum,enhanced the expression of liver regeneration-related genes HGF and Cyclin D1 in tissues,and inhibited the expression of liver regeneration termination gene TGF-?,increased the positive cells number of ki67 and the expression of PCNA protein.Moreover,there was no significant difference between the two intervention groups.(7)ADSC-CM component detection:VEGF,ANG-1,ANG-2,and b-FGF contained in miniature pigs ADSC-CM were detected by ELISA to promote angiogenesis.TNF-?,IL-1?,and IL-6 regulate inflammatory response and liver regeneration.In summary,both of ADSCs and ADSC-CM intervention via liver parenchyma transplantation can improve the histopathological damage and ultrastructural changes of liver ischemia-reperfusion combined with partial hepatectomy.It also can reduce excessive inflammatory response,oxidative stress response,reduce apoptosis,promote liver regeneration.This study successfully applied ADSCs and ADSC-CM to treat hepatic injury after hepatic ischemia-reperfusion combined with partial hepatectomy.ADSCs and ADSC-CM both have the functions of anti-oxidation,anti-inflammatory,anti-apoptosis and promoting liver regeneration.There is no significant difference between two groups.It is speculated that the mechanism of ADSCs functioning is probably through the paracrine pathway of secreting cytokines.