Study On The Efficacy Of Canine Adipose-derived Stem Cells Regulating Ferroptosis To Alleviate Pancreatic Injur

Canine acute pancreatitis(AP)is a multiple diseases with pancreatic edema,bleeding,necrosis,and inflammatory reaction as the main symptoms.The deterioration of the condition leads to systemic inflammatory syndrome and organ dysfunction.Adipose stem cells(ADSCs)are equipotent stem cells with self-renewal,multidirectional differentiation potential,easy acquisition,and high reproductive capacity,showing great potential in the treatment of diseases.However,its effect and mechanism in canine AP have not been clarified.Therefore,this study intends to explore the therapeutic effect and mechanism of canine ADSCs and adipose stem cellconditioned medium(ADSCS-CM)in AP,which is of great significance in improving the survival rate of dogs with AP.ADSCs were isolated and cultured from a canine sickle ligament in morphology,marker,and differentiation ability identification,and concentrated ADSCS-CM was prepared.ADSCs(1×106cells /kg)and ADSCS-CM(the same number of cells concentrated CM)were injected into the tail vein and the auricular vein of rats and rabbits,respectively,for pyrogens tumorigenesis monitoring.Twenty dogs were randomly divided into sham operation group(CON),AP model group(AP),ADSCs treatment group(ADSCs),and ADSCS-CM treatment group(CM).AP model was established by retrograde injection(5% carbocyclic sodium 0.1 m L/kg,trypsin 3500U/kg),and anesthesia monitoring was performed.ADSCs and ADSCS-CM were injected via a cephalic vein in the ADSCs group and CM group,and the same amount of normal saline was injected in the CON group and AP group.Physical examination,pain score,and B-ultrasound were performed 24 h postoperatively to examine the echo and morphology of the pancreas.Blood samples were collected for routine blood,biochemical,and blood gas monitoring,and compared with 5 cases of natural disease collected clinically,to explore the therapeutic effect of AP in dogs.And then,the histopathological changes were respected by HE staining,and the levels of oxidative stress in the pancreas and serum were detected by the kit.At last q RT-PCR and Western Blot were used to detect the Keap1-Nrf2 signaling pathway and iron-related genes in the pancreas to further clarify the mechanism of action.The results show:(1)Canine ADSCs were successfully prepared and the grew adherent and fusiform,expressing stem cell-specific antigens CD73,CD90 and CD105,but not CD34.ADSCs could differentiate into advocates and osteoblasts after induction in vitro.ADSCs and ADSCS-CM showed no pyrogenicity,tumorigenicity,or influence on organs after intravenous injection.(2)Serum lipase and amylase were significantly increased by retrograde injection of drugs into the pancreatic duct,and c PL was positive,which was consistent with the results of clinical natural disease cases.The postoperative pain score of the CON group was considerably lower than that of the other three groups.There were no significant differences in physical examination,introspective monitoring,blood routine,biochemical,and other blood gas indexes among all groups.The degree of bleeding,vascular degeneration,and inflammatory infiltration of pancreatic tissue in ADSCs and the CM group was less than that in the AP group.(3)Compared with the AP group,m RNA expressions of Keap1,Nrf2,and GPx4 in the pancreas of ADSCs and ADSCS-CM were significantly increased,and GST m RNA expression was significantly decreased after injection.The expression of FTH and GPx4 protein was significantly increased,while TFR protein was significantly decreased.The contents of MPO in the pancreas and T-NOS,i-NOS,and MPO in serum were significantly decreased,while the activities of T-AOC and GPx in the pancreas and T-SOD in serum were significantly increased.In conclusion,the canine AP model established by retrograde injection of 5% sodium taurocholate and trypsin mixed solution is stable and has similar clinical characteristics;ADSCs and ADSCs-CM from canine reduce pancreatic cell degeneration,necrosis,and inflammatory cells through the Keap1-Nrf2 pathway,decrease oxidative stress and iron death levels and thus alleviate pancreatic injury.This not only provides theoretical support for the clinical investigation and application of ADSCs and ADSCs-CM but also clarifies their mechanisms of action,which is important for the discovery of new therapeutic strategies to reduce the mortality of affected animals.