Fig 1.
Effects of hUMSCs-exo@Cyasterone on the proliferation and apoptosis of Dex-induced BMSCs.
1A: MTT analysis indicated that hUMSCs-exo@Cyasterone had minimal impact on BMSCs toxicity and could partially reverse their proliferation ability. 1B: hUMSCs-exo@Cyasterone groups demonstrated an enhancement in the proliferation ability of Dex-induced BMSCs. 1C: EDU staining (400×) was employed to assess the effect of hUMSCs-exo@Cyasterone on BMSC model proliferation. 1D: Bar chart of statistical results showed hUMSCs-exo@Cyasterone groups exhibited an improvement in BMSCs proliferation to a certain extent. 1E: Flow cytometry analysis revealed that hUMSCs-exo@Cyasterone could mitigate the apoptosis of BMSC model. 1F: Bar chart of statistical results showed hUMSCs-exo@Cyasterone groups demonstrated a reduction in the apoptosis level of BMSCs model to a certain extent. 1G: Western blot analysis depicted that hUMSCs-exo@Cyasterone groups reduced the expression levels of apoptotic proteins. 1H: The statistical results of the fold changes of CTSD, BID, Caspase-1 and Caspase-9. Here, CYA represents Cyasterone, EXO is short for hUMSCs-exo, and EXO@CYA signifies hUMSCs-exo@Cyasterone. Statistical significance between different groups is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001, *P < 0.001, n = 3.
Fig 2.
The effect of hUMSCs-exo@Cyasterone on lysosomal membrane permeability of Dex-induced BMSCs.
2A: Lysotracker Red staining was employed to assess the effect of hUMSCs-exo@Cyasterone on the lysosomal membrane permeability of BMSC models. It was observed that hUMSCs-exo@Cyasterone could reduce the permeability of lysosomal membrane to some extent (600×). 2B: AO staining was utilized to evaluate lysosomal membrane permeability in BMSCs. Confocal results demonstrated that hUMSCs-exo@Cyasterone could mitigate the change in lysosome permeability to a certain extent (600 × , with decreased green fluorescence and increased red fluorescence). Transfection with CTSD-WT weakened the protective effect of hUMSCs-exo@Cyasterone on lysosomal membrane permeability, whereas CTSD-N258A enhanced this protective effect.
Fig 3.
Effects of hUMSCs-exo@Cyasterone on the lysosomal localization of CTSD protein in Dex-induced BMSCs.
3A: Both CTSD and Lamp1 were significantly up-regulated in the BMSC model, with co-localization observed between CTSD and Lamp1 (600×). All groups treated with hUMSCs-exo@Cyasterone exhibited a decrease in the expression levels of CTSD and Lamp1, and inhibited the expression of CTSD in lysosomes, with hUMSCs-exo@Cyasterone showing the strongest inhibitory effect. 3B: CTSD demonstrated consistent changes in both lysosome and cytoplasm. 3C: The statistical results of the fold changes of CTSD in lysosome and cytoplasm. Here, CYA represents Cyasterone, EXO is short for hUMSCs-exo, and EXO@CYA signifies hUMSCs-exo@Cyasterone. Statistical significance between different groups is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001, *P < 0.001, n = 3.
Fig 4.
Dex promotes N-glycosylation of CTSD protein in BMSCs.
4A: Bioinformatics predicted two N-glycosylation modification sites in rat CTSD, resulting in three N-glycosylation modification mutants, namely N258A and N134A. 4B: Western blot analysis demonstrated that Tunicamycin reduced the molecular weight of CTSD to some extent. 4C: Western blot analysis showed a decrease in the molecular weight of mature CTSD after transfection with N-glycosylated mutants N134A, N258A, or N134/N258A of CTSD. Among them, the N258A mutant exerted the greatest influence on the molecular weight of mature CTSD. 4D: The statistical results of the fold change of mature CTSD compared with pro-CTSD. 4E: The statistical results of the fold change of mature CTSD in N134A, N258A and N134/N258A.
Fig 5.
CTSD-N258A enhances the effect of hUMSCs-exo@Cyasterone on the proliferation and apoptosis of Dex-induced BMSCs.
5A: MTT analysis indicated that CTSD-N258A is involved in hUMSCs-exo@Cyasterone-mediated protection. 5B: Edu staining (400×) was used to analyze BMSC proliferation, revealing that hUMSCs-exo@Cyasterone partially mitigated the proliferation of Dex-induced BMSCs. 5C: Bar chart of statistical results showed CTSD-N258A enhanced the proliferative protection of hUMSCs-exo@Cyasterone on BMSCs. 5D: The bar chart of the statistical results of the apoptosis rate of CTSD-N258A in BMSC model. 5E: Flow cytometry analysis demonstrated that N-glycosylated CTSD-N258A inhibited the apoptosis level of the BMSC model to a certain extent. 5F: Western blot analysis revealed that CTSD-N258A inhibited the expression of apoptotic proteins to a certain extent. 5G: The statistical results of the fold changes of the CTSD, BID, Caspase-1 and Caspase-9 in CTSD-N258A treated BMSC model. Here, CYA represents Cyasterone, EXO is short for hUMSCs-exo, and EXO@CYA signifies hUMSCs-exo@Cyasterone. In addition, 1: Dex + EXO@CYA, 2: Dex + EXO@CYA+Vector, 3: Dex + EXO@CYA + WT, 4: Dex + EXO@CYA + N258A. Statistical significance between different groups is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001, *P < 0.001, n = 3.
Fig 6.
CTSD-N258A enhances the protective effect of hUMSCs-exo@Cyasterone on lysosomal membrane permeability in Dex-induced BMSCs.
6A: Lysotracker Red staining was employed to analyze lysosomal membrane permeability in BMSCs. The results demonstrated that hUMSCs-exo@Cyasterone could reduce lysosomal membrane permeability to some extent. Furthermore, CTSD-N258A increased the protective effect of hUMSCs-exo@Cyasterone, as indicated by weakened red fluorescence (600×). 6B: AO staining was performed to assess lysosomal permeability in the BMSC model treated with hUMSCs-exo@Cyasterone. The results revealed decreased lysosomal membrane stability induced by Dex, accompanied by enhanced cytoplasmic green fluorescence (600×). Moreover, hUMSCs-exo@Cyasterone groups exhibited inhibition of lysosomal permeability to a certain extent.
Fig 7.
CTSD-N258A enhances the inhibitory effect of hUMSCs-exo@Cyasterone on the lysosomal localization of CTSD in Dex-induced BMSCs.
7A: The lysosomal localization of CTSD in the BMSC model was observed through immunofluorescence. Compared with the vector group, CTSD-WT transfection further promoted the expression of CTSD and Lamp1, with a certain degree of co-localization observed between CTSD and Lamp1. In contrast, transfection of CTSD-N258A resulted in decreased expression of CTSD and Lamp1 (600×). 7B: Western blot analysis revealed that transfection with CTSD-N258A decreased the expression of CTSD. 7C: The statistical results of the fold changes of the CTSD protein level in CTSD-N258A treated BMSC model in lysosome and cytoplasm. Here, CYA represents Cyasterone, EXO is short for hUMSCs-exo, and EXO@CYA signifies hUMSCs-exo@Cyasterone. In addition, 1: Dex + EXO@CYA, 2: Dex + EXO@CYA+Vector, 3: Dex + EXO@CYA + WT, 4: Dex + EXO@CYA + N258A. Significant differences between different groups are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001, *P < 0.001, n = 3.
Fig 8.
The graphic abstract of the protective effect of hUMSCs-exo@Cyasterone in Dex-induced BMSCs.
Dex promoted the lysosomal membrane permeability by up-regulating the pro-CTSD and m-CTSD, thus to increase the protein expression level of cytoplasmic CTSD and finally resulted in an improved apoptosis-related protein expression levels of BID, Caspase-1 and Caspase-9. However, hUMSCs-exo@Cyasterone ameliorated this pathological state. This graphic abstract showed hUMSCs-exo@Cyasterone alleviated the cellular model, the Dex-induced BMSCs, through the N-glycosylation modulation of CTSD (specifically CTSD-N258A). Here, m-CTSD is short for mature-CTSD.