Study On Protection Of Mesenchymal Stem Cells-Conditioned Medium Against 2,5-Hexanedione-Induced VSC4.1 Cell Autophagy And Its Mechanism

Objective: The purpose of this study is to investigate the mechanism of Mesenchymal stem cells-conditioned medium protects VSC4.1 cells against HD-induced autophagy Materials and Methods: Bone marrow mesenchymal(BMSCs)was extract from 3-4 weeks old SD rats.collectes the conditioned medium and concenrated at the ratio of 1:10.We use VSC4.1 cell for experient.Set up 5 experiement group :(1)blank control group: no treatment to the cells.(2)HD expose group: vsc4.1 cells were treated with 25 mM HD for 24 hours.(3)MSC-CM control group: after HD expose add 5%?10%?15% MSC-CM add into the medium and incubated for 24 h.(4)NGF group:after HD expose add 100ng/mL NGF to the medium and incubated to the medium.(5)Inhibitor group: incubated before or at the same time with MSC-CM.Cell viability was observed by MTT and LDH assay.The content of NGF in MSC-CM was detected by ELISA.LC3-DAPI was used to detced the autopgagy in VSC4.1 cells.Using electron microscopy to detect autophagosome and autolysosome in VSC4.1 cell.The exprission of LC3? p62?TrkA?p-TrkA?mTOR?p-mTOR?ULK?p-ULK was detcted by western blot.Result:1.Effect of BMSC-CM on autophagy of VSC4.1 cells exposed to HD Increased number of LC3-positive dots in HD group than control(P<0.05).With the increase of MSC-CM concentration,the number of LC3-positive dots decreased significantly and decreased in dose-dependent manner.(P<0.05).The number of autophagic vacuoles and the expression of LC3 II / I was decreased in MSC-CM group(P<0.05),and the expression of p62 protein was significantly increased(P<0.05)in theMSC-CM group.2.Effect of BMSC-CM on cell viability HD significantly decreased the cell viability and increased the cell death.The cell viability was increased with MSC-CM treatment and increased in dose-dependent manner.After add PIK-? to the cell,the cell death was decreased,same as the BMSC-CM.3.Level of NGF in BMSC-CM and expressions of TrkA and phosphorylated TrkA in VSC4.1 cells The concertration of NGF in MSC-CM was examined by ELISA.HD significantly decreased expression levels of p-TrkA in VSC4.1 cells than control(P<0.05),the expression level of p-TrkA was higher in BMSC-CM group than HD group(P<0.05)and increased in dose-dependent manner.4 NGF is a key regulator in BMSC-CM against HD-induced autophagy compare with MSC-CM group and NGF group,the number of LC3 positive dots and autophagic vacuoles was increased in anti-NGF group(P<0.05).The ratio of LC3 II / I was increased in anti-NGF group(P<0.05).The expression of p62 was decreased in anti-NGF group(P<0.05).5.MSC-CM affect the activity of PI3K/Akt/mTOR in VSC 4.1 cells HD decreased the level of p-Akt(P<0.05),the expression was recovered by BMSC-CM supplementation(P<0.05).Inhibitor group decreased the level of p-Akt compare with BMSC-CM group(P<0.05).6.MSC-CM affect the activity of ULK1 in VSC 4.1 cells HD decreased the level of p-mtor and increased the level of ULK1 than control(P<0.05),and the expression was recovered by BMSC-CM supplementation(P<0.05).Inhibitor group decreased the level of p-mtor and increased the level of ULK1(P<0.05).7.NGF activatesPI3K/Akt signaling pathway to regulate autophagy LC3-positive dots significant decreased in MSC-CM group compare with HD group(P<0.05),while LY294002 and Rapamycin interfered group significantly increased than MSC-CM group(P<0.05).Same as the autophagic vacuoles.The ratio of LC3 II / I was significantly lower in MSC-CM group than that in HD group(P<0.05),and in LY294002 and Rapamycin groups was significantly higher MSC-CM group(P<0.05).The expression of p62 was decreased in the MSC-CM than HD group(P<0.05),the expression was increased in inhibitor group than MSC-CM group(P<0.05).Conclusion: BMSC-CM inhibits HD-induced excessive autophagy in VSC4.1 cells.BMSC-CM protects against HD-induced autophagy in VSC4.1 cells via NGF-dependent activation of PI3K/Akt/mTOR pathway.