Higher senescence markers β-galactosidase and p16 ink4a were expressed in aging TSPCs.
It was reported in previous researches that aging TSPCs have a significantly higher p16 ink4a and β-galactosidase expression level[2, 14]. To confirm that we detected the expressions of p16 ink4a and β-galactosidase in young and aging TSPCs by western-blot. As is shown in Figure.1A, there were higher expressions of p16 ink4a and β-galactosidase in aging TSPCs.
The adipogenic differentiation was inhibited in aging TSPCs.
In order to explore whether the accumulation of adipocytes in aging tendon was caused by the change of adipogenic ability of TSPCs during aging, we conducted adipogenic induction culture of TSPCs in vitro. First of all, through oil red O staining, we intuitively found that the number of lipid droplets in aging TSPCs after adipogenic induction was significantly lower than that in young TSPCs (Fig. 1B), and the quantitative analysis (Fig. 1C) also showed that there was a significant difference in the number of lipid droplets in young and aging TSPCs. In qRT-PCR results, we found that Perilipin and FABP4 were significantly reduced in aging rat TSPCs after adipogenesis induction (Fig. 1D), while the corresponding content of Perilipin, C/EBPα and FABP4 in aging TSPCs were significantly reduced at the protein level (Fig. 1E&F). The similar results were shown by the Immunofluorescence staining (Fig. 1G).
RNA-seq analysis of gene expression profile of young and aging TSCs.
In order to analyze the potential causes of decreased adipogenic differentiation ability caused by aging in vitro, RNA sequencing was used to observe the changes of TSPCs expression profile. According to the results of heat map and volcanic map (Fig. 2A&B), there were 1653 genes with more than twice difference between young and aging TSPCs, among which 917 genes were up-regulated and 736 genes were down-regulated. Among the 10 pathways with the greatest differences between the younger and aging groups, KEGG results showed that lipid-related pathways included the ‘PPARγ signaling pathway’ (rno03320)and the ‘PI3K-Akt pathway’ (rno04151)(Fig. 2C).
PPAR γ signaling pathway was down-regulated in aging TPSCs.
In order to determine the exact pathway affecting the lipid formation of TSPCs in aging rats, we verified the expression of the pathway molecules according to the sequencing results, and found that the gene expression of molecules in the PPARγ signaling pathway, such as CD36, FABP3, OLR1, Perilipin, RXRA and Slc27a6 were significantly decreased (Fig. 3A). The results of western blot showed that the expression of PPARγ, Slc27a6, RXRA, angptl-4 and perilipin were lower in young(Y) TSPCs compared with aging(A) TSPCs (Fig. 3B). The qRT-PCR and western blot results demonstrated that the PPARγ signaling pathway was inhibited in the aging rat TSPCs.
The adipogenesis of aging TSCs was inhibited through downregulating PPAR γ signaling pathway.
To verify whether PPARγ pathway is the key influencing factors for aging TSPCs differentiating into adipocytes, we added the specificity agonist of PPARγ pathway Rosiglitazone maleate(RM) with 4 different concentrations(0.04, 0.4, 4, 40 µM), in the induction medium. Firstly, western blot was conducted to detect the expression of adipogenic related markers(PPARγ, C/EBPα, perilipin, FABP4). We found that the expression of PPARγ, C/EBPα, perilipin were elevated in a dose-dependent manner, and there was the highest expression with the concentration of 100 ng/ml of Rosiglitazone maleate(Fig. 4A). Then Oil red O staining and the qRT-PCR were conducted, it was revealed that after adding Rosiglitazone maleate agonist with the concentration of 100 ng/ml into the induction medium of aging TSPCs, there had been a marked increase in the number of lipid droplets (Fig. 4B- 1, 2, 3, 4). At the same time, The expression of PPARγ, perilipin and Slc27a6 (Fig. 4C-1) in the PPARγ signaling pathway in aging TSPCs were up-regulated compared with the aging TSPCs without Rosiglitazone maleate, though only the expression of PPARγ was significantly different (Fig. 4C-2).