Rab32 facilitates Schwann cell pyroptosis in rats following peripheral nerve injury by elevating ROS levels

Abstract

Background: Peripheral nerve injury (PNI) is commonly observed in clinical practice, yet the underlying mechanisms remain unclear. This study investigated the correlation between the expression of a Ras-related protein Rab32 and pyroptosis in rats following PNI, and potential mechanisms have been explored by which Rab32 may influence Schwann cells pyroptosis and ultimately peripheral nerve regeneration (PNR) through the regulation of Reactive oxygen species (ROS) levels.

Methods: The authors investigated the induction of Schwann cell pyroptosis and the elevated expression of Rab32 in a rat model of PNI. In vitro experiments revealed an upregulation of Rab32 during Schwann cell pyroptosis. Furthermore, the effect of Rab32 on the level of ROS in mitochondria in pyroptosis model has also been studied. Finally, the effects of knocking down the Rab32 gene on PNR were assessed, morphology, sensory and motor functions of sciatic nerves, electrophysiology and immunohistochemical analysis were conducted to assess the therapeutic efficacy.

Results: Silencing Rab32 attenuated PNI-induced Schwann cell pyroptosis and promoted peripheral nerve regeneration. Furthermore, our findings demonstrated that Rab32 induces significant oxidative stress by damaging the mitochondria of Schwann cells in the pyroptosis model in vitro.

Conclusion: Rab32 exacerbated Schwann cell pyroptosis in PNI model, leading to delayed peripheral nerve regeneration. Rab32 can be a potential target for future therapeutic strategy in the treatment of peripheral nerve injuries.

Keywords: Peripheral nerve regeneration; Pyroptosis; Rab32; Schwann cells.

Fig. 1

PNI induced pyroptosis and upregulated Rab32 expression. A Immunofluorescence staining for S100 (green) and GSDMD (red) in nerve tissues. Nuclei were labeled using DAPI (blue). Scale bar = 20 µm. B Heat map summarizing the DEGs related to Rab32 and pyroptosis-related gene. C The GO biological process enrichment analysis of DEGs. D The KEGG biological process enrichment analysis of DEGs. E–G Protein expression levels and quantitative results of Rab32 in Sham and PNI groups. (H) Immunohistochemical staining of Rab32 in peripheral nerve tissue. **P < 0.01. Scale bar = 50 µm

Fig. 2

Knocking down of Rab32 significantly reduces the mitochondrial ROS content in Schwann cells in vitro. A Western blotting accessed the expression of Rab32 after Schwann cells were infected with AAV. shRab32 for 24 h. Schwann cells were infected with AAV. shRab32 for a duration of 24 h, followed by induction of cellular pyroptosis using LPS and ATP. **p < 0.01 compared to the shRNA 2 group. *p < 0.05 compared to the shRNA 2 group. B and C Representative immunofluorescence images and quantitative analysis of the level of mitochondrial ROS using MitoSOX staining. Scale bar = 20 µm. **p < 0.01 compared to the LA/shRab32 group. *p < 0.05 compared to the LA/shRab32 group. D The representative images of mitochondrial membrane potential using JC-1 staining. Scale bar = 50 µm. E Quantification of the red-to-green (Percentage of control). Red: JC-1 monomers, green: JC-1 aggregates. **p < 0.01 compared to the LA group. *p < 0.05 compared to the LA group

Fig. 3

The impact of MitoQ on pyroptosis in vitro. A The level of ROS was assessed by DCFH-DA staining. B Expression of NLRP3 mRNA was analyzed by real-time PCR. C and D The levels of IL-1β and IL-18 in the culture medium were determined by ELISA. E The expression of Cleaved-cas1, N-GSDMD and NLRP3 protein was evaluated by western blotting, and representative bands are shown. F–H The histograms show the expression of the Cleaved-cas1, N-GSDMD and NLRP3 proteins in according to the semi-quantitative analysis of the bands. I Representative immunofluorescence images showing the location and level of NLRP3 in Schwann cells. J The quantitative analysis of mean fluorescence intensity (MFI) of NLRP3 in each group. Green: NLRP3, blue: DAPI. Scale bar = 20 µm. **p < 0.01, *p < 0.05

Fig. 4

Knockdown of Rab32 attenuates PNI-induced pyroptosis in vivo. Peripheral nerves tissue was infected with AAV.shRab32, followed by PNI treatment. These samples were harvested on the 3rd day postoperatively. A and B Expression of NLRP3 and Rab32 mRNA was analyzed by real-time PCR. C The expression of Cleaved-cas1, Rab32, N-GSDMD and NLRP3 protein was evaluated by western blotting, and representative bands are shown. D–G The histograms show the expression of the Rab32, N-GSDMD, Cleaved-cas1 and NLRP3 proteins in according to the semi-quantitative analysis of the bands. H Immunofluorescent analysis revealed the localization of NLPR3 in Schwann cell within the tissue of the peripheral nervous. Red: NLRP3, Green: S100 (Schwann cell marker), and bule: DAPI. Scale bar = 100 μM. **p < 0.01, *p < 0.05

Fig. 5

Silencing Rab32 improved the function of peripheral nerves in the postoperative period. The function of peripheral nerves had been evaluated during pre-surgery, and the 4th, 8th, and 12th postoperative weeks. A and B The representative image of footprints and quality analysis in Sciatic Functional Index (SFI) assay. C The relative level of Von Frey withdrawal threshold. D The representative image of electrophysiological analysis. E and F The histograms illustrated the levels of Latency of CMAP onset and peak amplitude of CMAP. **p < 0.01, *p < 0.05

Fig. 6

Rab32 downregulation improve promotes the regeneration of peripheral nerves in vivo. A TB staining examined the thickness of the myelin sheath in peripheral nerves at 12th postoperative week, Scale bar = 10 µm. TEM analysis evaluated regeneration of peripheral nerves at 6th and 12th postoperative week. Scale bar = 5 µm (upper), and 1 µm (lower). B Thickness of myelin sheath. C Number of myelinated axons. D Average myelinated axons diameter. E G-ratio. **p < 0.01, *p < 0.05

Fig. 7

Silencing Rab32 improve PNI induced Denervation drives Gastrocnemius muscle atrophy. The severity of denervation-induced gastrocnemius muscle atrophy was evaluated at 12 weeks postoperatively. A The gastrocnemius muscle was assessed for pathological changes through macroscopic analysis (upper) as well as H&E (middle) and Masson staining (lower). B–E The degree of recovery in atrophied gastrocnemius muscles were accessed by muscle weight (ratio injured/healthy limb), muscle fiber area, muscle fiber diameter and the collagen volume fraction (%), respectively. **p < 0.01, *p < 0.05. Scale bar = 40 µm

Fig. 8

Rab32 facilitates Schwann cell pyroptosis in rats following peripheral nerve injury by elevating ROS levels