Mechanisms Of Oxytocin Delaying Hepatocyte Senescence By Inducing Autophagy

Background and SignificanceClinical studies have found that the poor prognosis of elderly donors after liver transplantation is closely related to the poor quality of donor liver,and hepatocyte senescence is one of the main reasons leading to the poor quality of donor liver in the elderly.Aging can cause imbalance of tissue homeostasis and decrease of organ regeneration ability in mammals,mainly due to accumulation of cell damage caused by small-scale chronic stress.Autophagy plays an important role in the process of cell aging,and the decrease of autophagy level is one of the signs of aging.It is of great significance to explore the mechanism of liver aging,delay the senescence of hepatocyte,and promote the regeneration of hepatocyte to improve the quality of the elderly liver and delay the progress of many diseases related to the liver.Oxytocin(OT)is a hormone synthesized by supraoptic nucleus and paraventricular nucleus of hypothalamus and stored in neurohypophysis.However,in recent years,studies have found that besides the central nervous system,many peripheral organs such as the heart,uterus,thymus and alimentary can synthesize and release OT,and some cells express Oxytocin Receptor(OTR)on the surface.Recent studies have found that OT can promote myocardial repair after ischemia,differentiation and regeneration of fibroblast stem cells,skeletal muscle stem cells and adult hippocampal nerves.OT can restore muscle regeneration in aged mice by improving the function of muscle stem cells.Whether OTR also exists in liver and whether its activation can delay liver cell aging is still unknown.In this subject,we mainly study the expression and distribution characteristics of OTR in the liver and its role and mechanism in hepatocyte regeneration and slowing down hepatocyte senescence.We found that hepatocyte expressed OTR,and their expression levels gradually decreased with age in human liver,and their expression levels in liver of aged mice are also significantly lower than that of young mice.It was so consistent that OT levels in human and mouse serum also decreased significantly with age.In vitro experiments such as hepatocyte culture and mouse hepatocyte organoid experiments had found that OT could promote hepatocyte regeneration.Furthermore,through the research on the in vivo model of partial hepatectomy and CCl4-induced acute liver injury,it was found that OT promoted liver regeneration,relieved and promoted repair acute liver injury caused by CCl4 in aged mice.It was noteworthy that we found that OT promotes autophagy of hepatocytes,whether from in vitro or in vivo studies.The autophagy level of liver tissue in aged mice was obviously lower than that in young mice.OT significantly increased the level of autophagy in aged mice and had no significant effect on young mice,which was consistent with the results of OT in improving liver regeneration in aged mice.This finding revealed that OT delayed hepatocyte senescence by inducing autophagy,thus enhancing the function of elderly liver donors after liver transplantation and providing theoretical and practical basis for improving the quality of elderly liver donors.Methods1.Mouse hepatocyte strain:The cells used in the experiment were AML 12 cell(mouse hepatocyte),which was purchased from the stem cell bank of Chinese Academy of Sciences.The AML 12 cell was cultured in a DMEM/F-12 medium containing 10%FBS,1%ITS Liquid Media Supplement 100x),1%penicillin and streptomycin,and 40 ng/ml Dexamethasone.It needed to be cultured in a CO2 incubator at 37℃ and 5%CO2.After the cells were grown to a density of 70%-80%in a culture flask,cell experiments were performed.2.Mouse hepatocyte organoid model:C57BL/6 male mouse was used in this experiment.After euthanasia,the liver of the mouse was taken and the tissues were cut into pieces.Digested with DMEM/F-12 with 15 mM HEPSS digestive solution containing 0.3 mg/ml Collagenase Ⅱ(collagenase type Ⅱ)and 0.3 mg/ml Dispase Ⅱ.The process was carried out on a 37℃ constant temperature shaker for about 2h and 6 times.After 70μm cell strainer filtration,the filtrate was subjected to 37μm Reversible Strainer filtration and backwashed with cold Advanced DMEM/F-12.The filtrate containing the bile duct fragment was enriched by centrifugation at 290 g,℃,low speed horizontal refrigerated centrifuge.Finally,the collected bile duct fragments were uniformly mixed with Corning(?)Matrigel(?)Basement Membrane Matrix,50μl/well,and seeded in 24 well plates.After about 15min,Matrigel was coagulated,and then added with HepatiCultTM Organoid Growth Medium(Mouse),500μl/well.Put them in 37℃,5%CO2 primary incubator for culture.3.Animal model:First,the C57BL/6 male mice of 2months and 12months were used to construct a partial hepatectomy-hepatic regeneration model.They were randomly divided into two groups:normal saline group and OT group,with 6 mice in each group.OT treatment using subcutaneous injection.Secondly,the CCl4 acute liver injury model was constructed with 12months C57BL/6 male mice and randomly divided into four groups:Vehicle group(corn oil),CCl4 group(dissolve in corn oil),CCl4+OT(0.1 mg/kg)group and CCl4+OT(1 mg/kg)group.CCl4 was performed by intraperitoneal injection.OT was performed by subcutaneous injection.4.Cell model detection index:The treatment of cell was divided into three parts.The first part was to detect the effect of different concentrations of OT on the proliferation of AML 12 cell by CCK-8 kit.The second part was to detect the expression of OTR in AML 12 cell by Western Blot.The effects of different concentrations of OT on the expression of LC3Ⅱ/Ⅰ and Beclin-1 proteins in AML12 cells were detected both directly or after rapamycin-induced.In the third part,LC3 Lentivirus was transfected into AML12 cell,and immunofluorescence was used to detect the effect of different concentration OT on autophagy flow after rapamycin-induced.5.Mouse hepatocyte organoid model index:Different concentrations of OT treatment were used to detect the difference in the number and size of organoids compared with the control group during the Oh,12h,36h,and 72h time periods,and to take photos of 4 time periods.Western blot was used to detect the expression of OTR in organoids.6.Animal model detection index:In the partial hepatectomy model,after 3 days of liver resection,mouse liver tissue was taken,the weight of the remaining liver was weighed,data statistics were made,the difference in liver regeneration between OT treatment group and control group was calculated.In CCl4-induced acute liver injury model,HE staining was used to detect liver morphological changes of mice in different treatment groups.The levels of AST and ALT in serum of mice were determined by liver function test kit.Immunofluorescence technique was used to localize OTR in mouse liver.The expression of OTR in liver tissue was detected by Western Blot.The changes of LC3Ⅱ/Ⅰand Beclin-1 proteins in liver tissues of mice in different treatment groups were detected.Results1.Western blot showed that OTR was expressed in mouse liver tissue,AML12 cell and hepatocyte organoid.Immunofluorescence results showed that OTR was widely distributed in liver tissue,mainly on cell membrane.2.The expression of OTR was different in mice of different months’ages,and it decreased with the increase of age.And the expression of OTR in liver of human also decreased with the increase of age.Consistent with this,OT levels in human and mouse serum samples also decreased significantly with age.3.OT could promote the proliferation of AML 12 cell,and the effect was more obvious at 10-7Mol/L.OT promoted the growth of hepatocyte organoids in mice,and the effect was also significant at the concentration of 10"7 Mol/L.4.In the partial hepatectomy experiment of liver regeneration,OT significantly promoted the liver regeneration ability of mice at 12 months of age,but had no significant effect on mice of 2-3 months old.5.In the CCl4-induced acute liver injury model of 12months old mice,the damage degree of liver tissue in OT(0.1mg/kg)group was obviously weakened,the degree of inflammatory cell infiltration was reduced,the AST and ALT levels decreased significantly.And OT improved CC14-induced acute liver injury.6.It has the same expression characteristics as OTR,the levels of autophagy markers Beclin-1 and LC3Ⅱ/Ⅰ in liver of 2-3 months old and 24 months old mice decreased significantly with age.Compared with the control group,OT significantly increased the level of autophagy LC3Ⅱ/Ⅰ and promoted autophagy flow after OT was directly incubated or pretreated by rapamycin.In vivo experiments found that the autophagy level of liver tissue in aged mice with partial hepatectomy and CCl4-induced acute liver injury was significantly improved.This indicates that OT promotes hepatocyte regeneration and reverses hepatocyte aging by enhancing autophagy level.Conclusion1.OTR was expressed in hepatocytes,and its expression and serum OT levels decline with age in mice and human.2.OT can promote the proliferation of AML12 cell and the growth of hepatocyte organoids in mice3.OT can promote liver regeneration in aged mice,but has no effect on young mice.4.OT improves CCl4-induced acute liver injury.5.OT promotes hepatocyte autophagy.