Rejuvenation Of Senescent Hepatocytes In Aged Mice Via Hetrochronic Parabiosis

Cellular senescence,a stress-induced irreversible growth arrest,is often characterized by alteration of cell morphology and a reduction in physiological function.Senescent cells accumulate in various tissues and organs over time,which lead to the abnormalities of structure and function and an increase in the morbidity of various aged-related diseases.The liver is an organ with metabolic functions,and hepatocytes are the major parenchymal cells in the liver,which played a cardinal role in liver function such as bile synthesis,hepatin storage and lipid metabolism.A series of changes have taken place in the hepatic structure and function over time.Primary hepatic dysfunctions can be found in aged liver include increased hepatic volume,impaired proliferation capacity,the cavity appeared in nucleus,the lipofusion deposition in cytoplasmic,the abnormal DNA content and increased polyploidy hepatocytes.Due to the structural changes and functional abnormalities,the senescent hepatocytes are susceptible to suffering from various liver diseases in senility.For example,the elderly individuals are prone to hepatic fatty lesions,the ability of elderly mice declines to respond to chemical agents like CCl4,the level of liver inflammation increases and the development of liver fibrosis accelerates.It shows that the aging of hepatocytes is closely related to the development of liver diseases.So it is an inevitable requirement for the treatment of aged-related liver diseases to explore the way of hepatic rejuvenation.However,it is still unable to make an in-depth study of hepatic rejuvenation for the lack of liver aging animal models.In order to explore the effect of young blood environment in the process of senescent hepatocytes'rejuvenation,we established parabiosis models to realize the blood share between two mice,including isochronic youth-youth parabiosis(Iso-Y),elderly-elderly parabiosis(Iso-O)and heterochronic youth-elderly parabiosis(Het-Y,Het-O).What's more,the blood share is verified by fluorescence-activated cell sorting between two mice.Based on the establised parabiosis models,we analyzed the hepatocytes biological characteristics and physiological function respectively to evaluate the effect of young blood microenvironment in the rejuvenation of senescent hepatocytes.Compared to Iso-O,the percentages of SA-?-gal-positive hepatocytes decreased from 38.23%±1.86%to 4.84%±0.71%and the percentages of?-H2A.X-positive hepatocytes reduced from 53.98%±3.78%to 15.46%±1.86%.Moreover,the expression of cell cycle inhibitors such as P53,P21 and P16 decreased significantly and the inflammatory cytokines such as IL-1?,IL-6 and IL-8secreted by senescent hepatocytes also decreased after parabiosis.Polyploidy has been reported to increase with aging,and the polyploidy hepatocytes are susceptible to become senescent.Isolation the diploid,tetraploid and octoploid hepatocyte from Iso-Y,Iso-O,Het-Y and Het-O by fluorescence-activated cell sorting,which demonstrated that the ratio of octoploid decreased from 33.35%±0.85%to 15.97%±0.84%and the ratio of diploid hepatocytes increased from 7.41%±0.54%to 27.27%±0.71%compared with Iso-O.Furthermore,?-catenin and Hoechst 33342 were co-stained in the same liver section,which indicated the number of hepatocyte increased from 402±18 to 617±42 and the area of hepatocytes decreased from 645.99±32.72?m~2 to 356.93±22.33?m~2 compared with Iso-O in the same range of vision.Previously,impaired liver regeneration and delayed liver repopulation in hepatocytes transplantation of aged rodent have been reported in several studies.What's surprise is that young blood environment restored aging-induced suppression in hepatic proliferation.Both situ liver section staining and the primary hepatocytes culture in vitro indicated that the proliferative activity of senescent hepatocytes significantly strengthen under young blood environment compared with Iso-O.In conclusion,young blood environment reversed the senescence maker of hepatocytes in elderly mice and restored aged-dependent alterations in hepatic structure and proliferation function.Owing to the aging-induced alterations in both structure and function of senescent liver,chronic hepatitis,liver fibrosis and liver cancer are likely to occured in elderly individuals.With age,the lipid metabolism disorder appeared in hepatocytes,which significantly increased the level of hepatic fat accumulation and the morbility of liver fat lesions.However,it was found that the accumulation of fat in elderly mice was significantly alleviated under the regulation of young blood microenvironment.Compared to Iso-O mice,the positive area of Oil Red O reduced from 24.01%±3.48%to 4.15%±2.95%,and the positive area of Nile red was four times lower.Meanwhile,serological indicators also showed that TG,cholesterol,and LDL-c levels were significantly lower in aged mice(p<0.01),and the expression levels of ALT and AST were also decreased(p<0.01).According to the above results,we drew a conclusion that the young blood environment reversed age-dependent alterations in hepatic morphology,decreased the ratio of polyploidy hepatocytes and restored aging-induced suppression in hepatic proliferation.What's more,it could attenuate aging-induced hepatic steatosis and reduce aging-associated liver deterioration.Therefore,the study may inspire pharmacological strategies for reactivating the proliferative capacity of aged hepatocytes and treating aged-associated liver disease.It also provides a basis for understanding the relationship between aging and microenvironment and the mechanism of hepatic rejuvenation,which contributed to staving off the effect of hepatic senescence.