ADSCs Combined with Melatonin Promote Peripheral Nerve Regeneration through Autophagy

Abstract

Background: In the early stage of nerve injury, damaged tissue is cleared by autophagy. ADSCs can promote nerve axon regeneration. However, the microenvironment of the injury was changed, and ADSCs are easily apoptotic after transplantation. Mel plays a role in the apoptosis, proliferation, and differentiation of ADSCs. Therefore, we investigated whether Mel combined with ADSCs promoted peripheral nerve regeneration by enhancing early autophagy of injured nerves.

Materials and methods: SD rats were randomly split into the control group, model group, Mel group, ADSCs group, ADSCs + Mel group, and 3-MA group. On day 7, autophagy was observed and gait was detected on days 7, 14, 21, and 28. On the 28th day, the sciatic nerve of rats' renewal was detected.

Results: After 1 w, compare with the model group, the number of autophagosomes and lysosomes and the expressions of protein of LC3-II/LC3-I and Beclin-1 in the ADSCs + Mel group were prominently increased, while the 3-MA group was significantly decreased. After 4 w, the function of the sciatic nerve in ADSCs + Mel was similar to that in the control group. Compared with the model group, the ADSCs + Mel group significantly increased myelin regeneration and the number of motor neurons and reduced gastrocnemius atrophy.

Conclusions: It was confirmed that ADSCs combined with Mel could promote sciatic nerve regeneration in rats by changing the early autophagy activity of the injured sciatic nerve.

Figure 1

Drug allocation process.

Figure 2

ADSCs therapy process. (a)–(d) The sciatic nerve was crushed with tweezers. (e) The ADSCs were injected. (f) Postoperative suture. The lesion appears transparent (yellow arrows).

Figure 3

Grouping of experiments.

Figure 4

Characterization of adipose tissue-derived mesenchymal stem cells (ADSCs). (a) In passage three, the ADSCs exhibited a spindle-shaped fibroblastic morphology (magnification, ×100). (b) After dyeing with oil red O, the lipid droplets in the cells were stained red (magnification, ×400). (c) After staining with alizarin red dye, calcified nodules in cells were stained black (magnification, ×400).

Figure 5

Effects of ADSCs combined with Mel on functional recovery. (a) Recovery of foot morphology. (b) Rat footprint. (c) SFI scores for the functional recovery at different time points. The control group, ADSCs group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the model group, respectively. Each value is the mean ± SE of five independent determinations. ^##P < 0.01 and ^#p < 0.05, compared with the model group;  ^∗ ∗p < 0.05, the control group, ADSCs group, model group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the ADSCs + Mel group.

Figure 6

Expression of LC3 and Beclin-1 in rat sciatic nerve. (a) Western blot image of autophagy-related proteins LC3 and Beclin-1. (b) Beclin-1/ACTIN statistics. (c) LC3-II/LC3-I statistics. The control group, ADSCs group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the model group, respectively. Each value is the mean ± SE of four independent determinations. ^##P < 0.01, ^#p < 0.05 compared with the model group;  ^∗ ∗p < 0.05, the control group, ADSCs group, model group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the ADSCs + Mel group.

Figure 7

The mutations of autophagosomes on the 7th day after the sciatic nerve injury in rats were observed by electron microscopy. (a) Control group. (b) Model group. (c) ADSCs group. (d) Mel group. (e) Mel + ADSCs group. (f) 3-MA group. There are autophagosomes (yellow arrows) and lysosomes (red arrows) in the sciatic nerve. (g) Number of autophagosome statistics. The control group, ADSCs group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the model group, respectively. Each value is the mean ± SE of four independent determinations. ^##P < 0.01 and ^#p < 0.05 compared with the model group;  ^∗ ∗p < 0.05, the control group, ADSCs group, model group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the ADSCs + Mel group.

Figure 8

The expression of S-100 and NF-200 was detected by immunocytochemistry. (a) In experimental groups, the fluorescence intensity of NF-200 (green) and S-100 (red) elevated, which was detected by immunocytochemistry. (b)-(c) The fluorescence intensity. The control group, ADSCs group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the model group, respectively. Each value is the mean ± SE of five independent determinations. ^##P < 0.01 and ^#p < 0.05, compared with the model group;  ^∗ ∗p < 0.05, the control group, ADSCs group, model group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the ADSCs + Mel group; scale bar = 100 μm.

Figure 9

The immunofluorescence intensity expression of MBP and NF-200 was detected by immunocytochemistry. (a) In experimental groups, the fluorescence intensity of MBP (red) and NF-200 (green) elevated, which was detected by immunocytochemistry. (b)-(c) The fluorescence intensity was measured by integrated density pixel. The control group, ADSCs group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the model group, respectively. Each value is the mean ± SE of five independent determinations. ^##P < 0.01 and ^#p < 0.05, compared with the model group;  ^∗ ∗p < 0.05, the control group, ADSCs group, model group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the ADSCs + Mel group; scale bar = 100 μm.

Figure 10

The structure of sciatic nerve in different groups. (a) TEM staining of distal sciatic nerve injury in rats. Scale bar = 10 μm. (b) LFB staining of distal sciatic nerve injury in rats. Scale bar = 100 μm. (c) Myelin sheath thickness in each group. (d) Percentage of myelin sheath staining. The control group, ADSCs group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the model group, respectively. Each value is the mean ± SE of five independent determinations. ^##P < 0.01 and ^#p < 0.05, compared with the model the group;  ^∗ ∗p < 0.05, the control group, ADSCs group, model group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the ADSCs + Mel group.

Figure 11

Histological assessment of the gastrocnemius muscle. (a) Image of the normal and surgical side of the gastrocnemius, Masson trichromatic staining, and H&E staining of the cross-section of the gastrocnemius. Scale bar = 50 μm. (b) Gastrocnemius wet weight ratio. (c) The average percentage of collagen fiber area. (d) Muscle fiber cross-sectional area statistical results. The control group, ADSCs group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the model group, respectively. Each value is the mean ± SE of five independent determinations. ^##P < 0.01 and ^#p < 0.05 compared with the model group;  ^∗ ∗p < 0.05, the control group, ADSCs group, model group, Mel group, ADSCs + Mel group, and 3-MA group were compared with the ADSCs + Mel group; scale bar = 20 μm.

Figure 12

Histological assessment of the spinal cord. (a) L4 ventral horn Nissl staining. (b) The number of Nissl bodies in each group. Each value is the mean ± SE of five independent determinations. ^##P < 0.01, compared with the model group;  ^∗ ∗p < 0.05, compared with the ADSCs + Mel group.