ROS scavengers MitoQ inhibit pyroptosis in Schwann cells
The mitochondrial ROS scavenger, MitoQ, was used to examine the impact of mitochondrial ROS levels on the level of pyroptosis in Schwann cells. The DCFH-DA tests (Fig. 3A) shows that MitoQ (M) effectively reduces the ROS level in Schwann cells treated with LPS/ATP (LA). The RT-PCR results showed that the gene expression of NLRP3 (Fig. 3B) in the LA group was significantly downregulated compared with the LA group, although they were slightly higher than those in the Ctrl and Ctrl/M groups. Furthermore, the enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of IL-1β (Fig. 3C) and IL-18 (Fig. 3D) in each group, which allowed for the evaluation of the pyroptosis level of Schwann cells in each group, the results showed that the IL18 and IL1β levels in the LA/M group were significantly lower than those in the LA group (p < 0.05). Subsequently, Western blotting was used to assess the levels of pyroptosis related proteins Cleaved-cas1, N-GSDMD and NLRP3 in each group. The results showed that the levels of these proteins in the LA/M group were significantly lower than those in the LA group, but still higher than those in the Ctrl group and Ctrl/M group (Fig. 3E–H) (p < 0.05). Finally, immunofluorescence staining was performed to determine the localization of NLRP3, a specific marker for pyroptosis (Fig. 3I). The mean fluorescence intensity (MFI) of NLRP3 was significantly lower in the LA/M group group compared to the LA group (Fig. 3J) (p < 0.05). These results provide evidence that the application of MitoQ significantly inhibits pyroptosis in Schwann cells in vitro.
Rab32 involved in PNI-induced pyroptosis in vivo.
After pyroptosis, a large amount of inflammatory substances are released, which hinders the regeneration of surrounding nerves. Therefore, reducing the level of pyroptosis in the tissue is of great significance for the regeneration of peripheral nerves, especially Schwann cells pyroptosis. We first verified the effectiveness of AAV in vivo to knock down levels of Rab32. The results showed that the Rab32 gene (Fig. 4A) and protein levels (Fig. 4D) in the PNI/ShRab32 group were significantly lower than in the PNI group, indicating successful knockdown of Rab32 in the PNI/ ShRab32 group. The RT-PCR results showed that the gene expression of NLRP3 in the PNI/ShRab32 group was significantly downregulated compared with the PNI group, although it was slightly higher than those in the Sham and Sham/ShRab32 groups (Fig. 4B). The subsequent Western blotting results (Fig. 4C) show that the levels of pyroptosis-related protein, Cleaved-cas1, N-GSDMD and NLRP3 were significantly reduced in the PNI/ShRab32 group compared with those in the PNI group, which were still higher than those in the Sham and Sham/ShRab32 groups, indicating that the reduction in Rab32 protein levels alleviates the level of pyroptosis in vivo (Fig. 4E–G). In addition, immunofluorescence staining was used to detect the localization of the specific pyroptosis marker, NLRP3, and specific Schwann cell marker, S100. The MFI of NLRP3 in the PNI/ShRab32 group was significantly lower than that in the PNI group, and this is consistent with the previous results (Fig. 4H).
Rab32 negatively regulates peripheral nerve function in the post-operative period.
To investigate the impact of Rab32 protein on peripheral nerve regeneration, we first used adeno-associated virus to knock down the levels of Rab32 in vivo. Then, we observed its effect on the regeneration of the rat sciatic nerve injury.
In order to evaluate the potential effects of Rab32 on the recovery of motor function in rats with sciatic nerve injury, we assessed the recovery of motor function by measuring SFI at 4, 8, and 12 weeks post-surgery (Fig. 5A and B). The results showed that there was no significant difference between the PNI group and the PNI/shRab32 group at the 4th week post-surgery, which is consistent with previous findings. This lack of difference might be due to the short duration of nerve repair initiation at this stage. However, at the 8th and 12th week, the SFI results in the PNI/shRab32 group were significantly better than those in the PNI group, indicating that downregulation of Rab32 levels can promote the recovery of sciatic nerve motor function.
The mechanical sensitivity test (Von Frey test) is a test that applies thin calibrated plastic filaments to the plantar surface of the hind paws. Different thicknesses or stiffnesses of Von Frey filaments are used to determine the threshold for eliciting withdrawal responses. In the study, the Von Frey test is used to evaluate the functional recovery after nerve crush at 4, 8, and 12 weeks post-surgery (Fig. 5C). No significant differences were observed at week 4, but the Von Frey test showed that the rats in the PNI/shRab32 group had faster functional recovery at 8 and 12 weeks post-surgery compared to the PNI group.
Electrophysiological tests are key indicators for evaluating recovery after sciatic nerve injury. In our study, electrophysiological indicators were evaluated at 4, 8, and 12 weeks post-surgery (Fig. 5D). The results showed that at the 8th and 12th weeks post-surgery, the CMAP latency and nerve conduction velocity indicators of the PNI/shRab32 group were significantly better than those of the PNI group (Fig. 5E and F), confirming that knocking down Rab32 levels can promote peripheral nerve regeneration.
Rab32 delayed promotes the regeneration of peripheral nerves in vivo.
Toluidine blue (TB) staining and TEM were used to evaluate remyelination of the regenerated nerves (Fig. 6A). According to the TB staining results, the myelin sheath thickness in the PNI/shRab32 group is significantly better than that in the PNI group, but worse than that in the Sham group and Sham/shRab32 group. The neuro-electron microscopy results at 6 and 12 weeks were evaluated. The findings indicate that the thickness of myelin sheath, number of myelinated axons, and myelinated axon diameter in the PNI/shRab32 group were superior to those in the PNI group (Fig. 6B-D), while they were still lower than those in the Sham group and Sham/shRab32 group (p < 0.05).
The G-ratio serves as an indicator of the ratio between neural axons and myelin sheath. A lower G-ratio value implies a greater abundance of myelin sheath, which facilitates efficient nerve regeneration. In this study, the results demonstrate significantly lower G-ratio values in the PNI/shRab32 group compared to the PNI group (p < 0.05) (Fig. 6E). Consequently, it can be inferred that decreased Rab32 protein levels contribute to increased myelin sheath formation, ultimately facilitating the regeneration of peripheral nerves.