Bone marrow mesenchymal stem cells protect against n-hexane-induced neuropathy through beclin 1-independent inhibition of autophagy

Abstract

Chronic exposure to n-hexane, a widely used organic solvent in industry, induces central-peripheral neuropathy, which is mediated by its active metabolite, 2,5-hexanedione (HD). We recently reported that transplantation of bone marrow-mesenchymal stem cells (BMSC) significantly ameliorated HD-induced neuronal damage and motor deficits in rats. However, the mechanisms remain unclear. Here, we reported that inhibition of HD-induced autophagy contributed to BMSC-afforded protection. BMSC transplantation significantly reduced the levels of microtubule-associated protein 1 light chain 3-II (LC3-II) and the degradation of sequestosome-1 (p62) in the spinal cord and sciatic nerve of HD-intoxicated rats. Downregulation of autophagy by BMSC was also confirmed in VSC4.1 cells exposed to HD. Moreover, inhibition of autophagy by PIK III mitigated the neurotoxic effects of HD and, meanwhile, abolished BMSC-afforded neuroprotection. Furthermore, we found that BMSC failed to interfere with Beclin 1, but promoted activation of mammalian target of rapamycin (mTOR). Unc-like kinse 1 (ULK1) was further recognized as the downstream target of mTOR responsible for BMSC-mediated inhibition of autophagy. Altogether, BMSC transplantation potently ameliorated HD-induced autophagy through beclin 1-independent activation of mTOR pathway, providing a novel insight for the therapeutic effects of BMSC against n-hexane and other environmental toxicants-induced neurotoxicity.

Figure 1

BMSC attenuates HD-induced activation of autophagy in the spinal cord and sciatic nerve of rats. (A,B) The level of LC3II (A) and p62 (B) was determined in both spinal cord and sciatic nerve of rats by western blot and the density of blots was quantified (the full-length gels were shown in Supplementary Figure S2A–D). (C) LC3 was stained in both spinal cord and sciatic nerve of rats and the representative images were shown. (D) MAP-2 and LC3 were co-stained in the spinal cord. White arrows represented MAP-2⁺/LC3⁺ cells in the fields. (E) Inhibition of autophagy by PIK III protects against HD-induced neurotoxicity and, meanwhile, blocks the neuroprotective effects of BMSC. VSC4.1 cells were treated with saline or HD (25 mM) for 24 h and then were treated with BMSC-CM (15%, v/v) or PIK III (20 uM) for additional 24 h. LDH analysis was used to detect the viability of VSC4.1 cell. (F) The level of LC3 II in VSC4.1 cells was determined in different groups and the density of blots was quantified (the full-length gels were shown in Supplementary Figure 2E). Quantified data are shown as mean ± sd. ^ap < 0.05, compared with control group; ^bp < 0.05, compared with HD group.

Figure 2

NGF is a key to mediate BMSC-afforded neuroprotection. (A) The protein levels of NGF were detected in the spinal cord and sciatic nerve of rats by using commercial ELISA kit. (B) Neutralization of NGF by using anti-NGF antibody abolishes the inhibitory effects of BMSC against HD-induced elevation of LC3II. VSC4.1 cells were treated with HD (25 mM) or saline for 24 h and then were treated with BMSC-CM (15%, v/v) or NGF (100 μM) in the presence or absence of anti-NGF antibody (10 μM) for additional 24 h. The levels of LC3 determined by western blot and the density of blots were quantified (the full-length gels were shown in Supplementary Figure S3A). (C) LC3 was stained in different groups and the representative images were shown. (D) TEM was used to detect the autophagosome in different groups. (E) K252a, the inhibitor of TrkA, attenuates the inhibitory effects of BMSC against HD-induced elevation of LC3II. VSC4.1 cells were treated with HD (25 mM) or saline for 24 h and then were treated with BMSC-CM (15%, v/v) with or without 1 h pre-treatment of the inhibitor of TrkA, K252a, for additional 24 h. The levels of LC3 determined by western blot and the density of blots were quantified (the full-length gels were shown in Supplementary Figure S3B). ^ap < 0.05, compared with control group; ^bp < 0.05, compared with HD group; ^cp < 0.05, compared with BMSC-CM group.

Figure 3

BMSC fails to interfere with the expression of Beclin 1, but stimulates activation of mTOR pathway. (A) The protein levels of Beclin 1 were determined in the spinal cord and sciatic nerve of rats by western blot and the density of blots were quantified (the full-length gels were shown in Supplementary Figure S4A,B). (B) The protein levels of mTOR and p-mTOR was determined in the spinal cord and sciatic nerve of rats by western blot and the density of blots were quantified (the full-length gels were shown in Supplementary Figure S4C,D). Quantified data are shown as mean ± SD. ^ap < 0.05, compared with control group; ^bp < 0.05, compared with HD group. (C) The inhibitors of mTOR (Rapamycin, Rap) attenuate the inhibitory effects of BMSC against HD-induced elevation of LC3II. VSC4.1 cells were treated with HD (25 mM) or saline for 24 h and then were treated with BMSC-CM (15%, v/v) in the presence or absence of Rap for additional 24 h. The levels of LC3 were determined by western blot and the density of blots was quantified (the full-length gels were shown in Supplementary Figure S4E). (D) Rap attenuates the inhibitory effects of BMSC against HD-induced degradation of p62 in VSC4.1 cells (the full-length gels were shown in Supplementary Figure S4F). (E) LC3II was staining in different groups and the representative images were shown. (F) TEM was performed in different groups and the representative images were shown. Data represent mean ± SD. ^ap < 0.05, compared with control group; ^bp < 0.05, compared with HD group; ^cp < 0.05, compared with BMSC-CM group.

Figure 4

mTOR-dependent activation of ULK1 contributes the anti-autophagic effects of BMSC. (A) The effects of BMSC transplantation on the levels of ULK1 and p-ULK-1 in the spinal cord and sciatic nerve of HD-intoxicated rats were detected using western blot and the density of blots was quantified (the full-length gels were shown in Supplementary Figure S5A,B). (B) Rapamycin abolished the inhibitory effects of BMSC against HD-induced phosphorylation of ULK1. VSC4.1 cells were treated with HD (25 mM) or saline for 24 h and then were treated with BMSC-CM (15%, v/v) in the presence or absence of Rap for additional 24 h. The levels of ULK1 and p-ULK-1 were determined by Western blot and the density of blots was quantified (the full-length gels were shown in Supplementary Figure S4C). ^ap < 0.05, compared with control group; ^bp < 0.05, compared with HD group; ^cp < 0.05, compared with BMSC-CM group.

Figure 5

Schematic diagram of experimental design. (A) The schematic diagram of experimental design about rats. After 7-day acclimatization, fifty rats were randomly divided into 5 groups (n = 10 for each group). Rats were treated with HD (400 mg/kg, i.p) or saline for 5 consecutive weeks (five times per week) and then received BMSC or saline by tail vein injection (additional 5 weeks later, rats were killed). (B) The schematic diagram of experimental design about VSC4.1 cells. Prepared VSC4.1 Cells were treated with HD (25 mM) or saline for 24 h and then were treated with BMSC-CM (15%, v/v) in the presence or absence of some pathway inhibitors or activators.