The Mechanism Of Heterochronic Parabiosis Effecting On The Knee Joint Cartilage Degeneration In Old Mice

Research BackgroundHeterochronic parabiosis can bring young blood components to old body by establishing a joint circulatory system via connecting the aged mouse circulation system with the young circulation system.Many studies have demonstrated that the model can improve the aged mouse tissues and organs(such as heart,brain,muscle,etc.)by changing its micro environment,and restore its regeneration function.Studies have suggested that GDF11 �young factor� is a critical factor in the aging process.Osteoarthritis(OA)is one of most common disability in aging people.However,it is unknown whether the model and GDF11 will affect the joint that relates to OA cartilage degeneration.In this study,we will observe the effect of heterochronic parabiosis mouse model on the cartilage metabolism in mice,verify the effect of GDF11 on the degeneration of cartilage tissue and its surrounding tissues,and use the heterochronic parabiosis mouse model to explore if there are other unknown "young factors" that are involved in the aging cartilage degeneration by analyzing their serum specimens using proteomics approach.Objective The purpose of this study was to observe the heterochronic parabiosis model effectting on knee joint cartilage in old mice.The effect of GDF11 on the degeneration of cartilage tissue and its surrounding tissues was verified.And the serum samples from heterochronic parabiosis model were collected in order to find younger factors by using proteomics.Methods 1 Mice differing in age were randomly divided into three groups-group 1,Heterochronic parabiosis(2-month-old/12-month-old,i.e.Young/Old)as the experimental group(Y/O group);group 2,Isochronic parabiosis(12 month old/12 month old,i.e.Old/Old)as the surgery control(O/O group);and group 3,12-month-old alone(12 monthold,Old)as the ageing control(O group).Old knee cartilages collected from all three groups at the time of 4 months post-surgery were compared.Fluorescence molecular tomography(FMT)was used to confirm whether the two mice in parabiosis shared a common blood circulation at the time of 2 weeks post-surgery.The knee joints of Old mice were examined using X-ray at the time of 4 months post-surgery.Histological scoring was assigned to grade the severity of osteoarthritis(OA).Immunohistochemistry and qRT-PCR were used to evaluate OA-related protein expression and gene expression,and chondrocyte proliferation was determined with EdU staining.2 The effect of GDF11 on cartilage metabolism was observed in vitro and in vivo.(1)In vitro: The chondrocytes were extracted and cultured from the knee joint cartilage after artificial knee replacement.rGDF11 were given in experimental group and only saline were given in control group.After chondrocytes being cultured 24 hours,several tests were conducted.Crystal Violet Staining was used to observe the morphology of chondrocytes.Chondrocytes proliferation was determined with EdU staining and scratch assay.Western blot and qRT-PCR were used to evaluate Smad2/3 signaling pathway related protein expression and gene expression and OA-related protein expression and gene expression.(2)In vivo: Old mice were randomly divided into two groups-group1,experimental group(rGDF11);group 2,control group(saline).Intraperitoneal injection was given in these two groups for 16 weeks seriesly.The knee joints of Old mice were examined using X-ray at the time of 4 months post-injection.Histological scoring was assigned to grade the severity of osteoarthritis(OA).Immunohistochemistry and qRT-PCR were used to evaluate OA-related protein expression and gene expression,and chondrocyte proliferation was determined with EdU staining.3.IBT proteomics: group 1,Heterochronic parabiosis(2-month-old/12-month-old,i.e.Young/Old)as the experimental group(Y/O group);group 2,Isochronic parabiosis(12month old/12 month old,i.e.Old/Old)as the control group(O/O group).The serum of old mice from group1 and group2 was collected for IBT proteomics in order to find more�young factors�.Results 1 The data from FMT imaging confirmed cross-circulation in the parabiotic pairs.Osteophytosis scores and Osteosclerosis scores in old cartilage from the Y/O group were significantly higher compared to the O/O and O groups(P<0.05).Loss of cartilage proteoglycan(PG)and OARIS in old mice from the Y/O group were significantly lower compared to O/O and O groups(P<0.05 for both).There was a statistically higher in the mRNA expression of collagen type II(Col2)and sex-determining region Y box 9(Sox9)in old cartilage from the Y/O group compared to those in the O/O and O groups(P<0.05 for both).On the contrary,in the Y/O group,mRNA expression of runt-related transcription factor 2(Runx2),collagen type X(Col10),and matrix metalloproteinase 13(MMP13)in old cartilage were significantly increased compared to the O/O and O groups(P<0.05 for both).The changes in Col2,Col10,MMP13,Runx2 and Sox9 at the protein level mimicked the alterations found at the mRNA level.The percentage of EdU-positive chondrocytes in young mice from the Y/O group was significantly higher compared to those of the O/O and O groups(P<0.05 for both).2 The effect of GDF11 on cartilage metabolism was observed in vitro and in vivo.(1)In vitro: The percentage of EdU-positive chondrocytes in the experimental group was significantly higher compared to those of the control group(P<0.05).There was a statistically higher in the mRNA expression of Col2 in old cartilage from the experimental group compared to those in the control groups(P<0.05 for both).There were no statistically significance in changes of Smad2 and Smad3 between two groups(P>0.05).But,the pSmad2 and pSmad3 at the protein levels in chondrocytes from the experimental group were higher than from the control group.The changes in Col2,Col10 and MMP13 at the protein level mimicked the alterations found at the mRNA level.(2)Osteophytosis scores and Osteosclerosis scores in old cartilage in the experimental group were significantly higher compared to the control group(P<0.05).Loss of cartilage proteoglycan(PG)and OARIS in old mice from the experimental group were significantly lower compared to the control group(P<0.05 for both).There was a statistically higher in the mRNA expression of collagen type II(Col2)in old cartilage from the experimental group compared to those in the control group(P<0.05 for both).On the contrary,in the experimental group,mRNA expression of Col10 and MMP13 in old cartilage was significantly decreased compared to the control group(P<0.05).There were no statistically significance in changes of Smad2 and Smad3 between two groups(P>0.05).The changes in Smad2/3,Col2,Col10,MMP13 at the protein level mimicked the alterations found at the mRNA level.The percentage of EdU-positive chondrocytes in young mice from the Y/O group was significantly higher compared to those of the O/O and O groups(P<0.05 for both).3.IBT proteomics: 379 significant different proteins were found in old serum from heterochronic parabiosis model.There were 285 up-regulating proteins and 94 down-regulating proteins among 379 significant different proteins.Conclusion 1 Heterochronic parabiosis exerts a positive effect on chondrocyte proliferation in the knee cartilage of old mice.2 GDF11 promotes the secretion of Col2 in chondrocytes by up-regulate Smad2/3,and GDF11 has a positive effect on chondrocyte proliferation in the knee cartilage of old mice.3 More �young factors� are found in old serum from heterochronic parabiosis model.