SS-31 promotes functional recovery after SCI
To evaluate the neuroprotective effect of SS-31 in mice with spinal cord contusions, we performed tissue staining and motor function evaluation. Synapsin (Syn) shows synaptic connectivity changes, the number of which can reflect motor cortical or spinal plasticity after traumatic injury [46]. A decrease in MAP2 in motoneurons distal to the lesion site indicates cytoskeletal abnormalities [47]. At 28 dpi, IF showed decreased MAP2 expression and fewer Syn-positive synapses on neurons in the SCI group than on neurons in the Sham group. The SCI mice treated with SS-31 had higher levels of neuronal MAP2 expression and more Syn-positive synapses than the SCI mice without any treatment (Fig. 1A–D). Additionally, the injured spinal cord lesion area was evaluated using HE and Masson staining, which revealed a glial scar area that was significantly expanded in the SCI group compared to the Sham group. However, SS-31 reduced the glial scar area in comparison to that in the SCI group (Fig. 1E, F). To further investigate the contribution of SS-31 to locomotive functional recovery, we performed footprint analysis, the inclined plane test and BMS score. Footprint analysis at 28 dpi showed significant gait recovery based on hind limb function in the SS-31+SCI group; in contrast, the mice in the SCI group remained unable to raise their hind limbs (Fig. 1G). The quantitative data measured by footprint analysis, including toe dragging and stride length, showed the same trends (Fig. 1H, I). Next, compared with the SCI group, the group of SS-31-treated SCI mice showed significantly higher BMS score and inclined plane angles at 14 dpi, 21 dpi and 28 dpi (Fig. 1J, K). In addition, we performed further experiments to determine the effects of SS-31 treatment in sham mice. As shown in Additional file 1: Fig. S1A–I, the MAP2 density, Syn numbers, toe dragging, stride length and BMS score exhibited no difference between non-SCI mice treated with and without SS-31. These results indicated that SS-31 treatment did not affect histological morphology and motor function recovery in non-SCI mice but effectively promoted functional recovery in SCI mice.
SS-31 attenuates pyroptosis after SCI
After traumatic tissue injury, inflammation is unavoidable. However, inflammation is a double-edged sword: rapid resolution of inflammation is necessary to ensure effective host defence and appropriate cell repair following an injury, but excessive inflammation induces further cell death, aggravating the injury [48]. A proinflammatory type of regulated cell death known as pyroptosis is mediated by cysteine-dependent aspartate-specific inflammatory proteases [48]. Pyroptosis has recently attracted considerable attention in relation to its specific function in inflammation. To determine whether SS-31 inhibits pyroptosis, we examined Caspase-1, GSDMD-N, NLRP3, NLRP1, ASC, IL-1β, and IL-18, which are essential for pyroptosis. IF staining of the SCI group revealed that Caspase-1 and NLRP3 fluorescence signals were strong in neurons. The SCI+SS-31 group, on the other hand, showed modest fluorescence signals (Fig. 2A–D). Notably, extensive evidence indicates that microglia-mediated pyroptosis is also a significant cause of neuroinflammation after SCI [13, 49]. Through IF staining, the numbers of Caspase-1- and NLRP3-positive microglial cells were detected after SCI (Additional file 1: Fig. S2A–D). Significant reductions in Caspase-1 and NLRP3 expression in neurons and microglia after SS-31 treatment indicated that SS-31 depressed pyroptosis following SCI. WB assessment was used to determine the expression levels of Caspase-1, GSDMD-N, NLRP3, NLRP1, ASC, IL-1β and IL-18. These pyroptosis-related proteins were substantially more abundant in the SCI group than in the Sham group. SS-31, nevertheless, reduced the expression of all seven proteins (Fig. 2E, F). As a result of these outcomes, SS-31 decreased pyroptosis after SCI.
SS-31 enhances autophagy after SCI
Increased autophagy has been observed after SCI, and increasing amounts of research have shown that autophagy can function as a prosurvival mechanism by controlling neural cell death to provide neuroprotection [50]. Additionally, previous research has demonstrated that SS-31 promotes autophagy in neurodegenerative diseases [23]. To test the effect of SS-31 on neural autophagy activity after SCI, we examined the protein levels of autophagosomal proteins (Beclin-1, VPS34, and LC3), lysosomal biogenesis-associated biomarkers (ATP6V1B2 and LAMP1), and autophagy substrate proteins (SQSTM1/p62) 3 days after SCI. As shown in Fig. 3A, B, the numbers of LC3 II puncta in each neuron increased following SCI and further increased after additional SS-31 treatment. Furthermore, following treatment with SS-31, the expression of p62 in neurons within the spinal cord lesion was decreased in comparison with that in the SCI group (Fig. 3C, D). As a substrate of autophagy, the SQSTM1/p62 protein is affected not only by autophagic flux, but also by the corresponding gene expression level. Therefore, we detected the mRNA level of Sqstm1 following SS-31 treatment in both Sham and SCI mice. After SCI, both the mRNA and protein levels of p62 were increased, and the protein level was further increased (Fig. 3E–G), indicating that impairment of autophagic flux occurred after SCI. Moreover, after treatment with SS-31, the mRNA level of Sqstm1 increased, while the protein level decreased (Fig. 3E–G), which indicated that SS-31 enhanced autophagic flux in SCI mice. Later, we detected the protein levels of autophagy-related genes. WB demonstrated that following SS-31 treatment, the levels of VPS34, Beclin-1, and LC3 II increased, while the level of p62 decreased. Nevertheless, the expression of ATP6V1B2 and LAMP1 did not differ among the three groups (Fig. 3F, G). Overall, these outcomes demonstrated that SCI resulted in impaired autophagy/autophagic flux and that SS-31 alleviated the disruption of autophagic flux.
The autophagy-activating, pyroptosis-inhibiting and functional recovery-promoting effects of SS-31 are inhibited by CQ
To further confirm that the beneficial effects of SS-31 on SCI recovery are due to an increase in autophagic activities, CQ, a classic autophagic flux inhibitor that inhibits lysosomal acidification [51], was used in subsequent research. As the WB and IF results in Fig. 4A–E revealed, the protein levels of VPS34 and Beclin-1 showed no change in the Sham and Sham+CQ groups, while LC3 II and p62 were upregulated in the Sham+CQ group. The results indicated that CQ did not affect the initiation of autophagy, but inhibited the activity of lysosomes and the fusion of lysosomes and autophagosomes, thus inhibiting autophagic flux in non-SCI mice. Then, IF demonstrated that the SCI+SS-31+CQ group had more LC3 II puncta in neurons than the SCI+SS-31 group. IF also showed that p62 levels in neurons were higher in SCI+SS-31+CQ mice than in SCI+SS-31 mice (Fig. 4A–C). The findings indicated that in SCI mice, CQ blocked autophagic flux, which was restored by SS-31. We employed WB to examine autophagy-related proteins in order to determine how CQ affected SS-31 therapy. WB demonstrated that the protein expression of Beclin-1 and VPS34 did not differ significantly between the SCI+SS-31 and SCI+SS-31+CQ groups (Fig. 4D, E). This demonstrated that CQ had no impact on autophagosome recruitment in SCI mice following SS-31 administration. WB also revealed that the LC3 II protein level was increased in the SCI+SS-31+CQ group in comparison with the SCI+SS-31 group. Furthermore, the level of p62 in SCI+SS-31+CQ mice was much greater than that in SCI+SS-31 mice (Fig. 4D, E). All of these investigations indicated that CQ inhibited SS-31’s efficient autophagy enhancement by blocking autophagic flux. Pyroptosis and autophagy are two distinct biological processes that determine cell fates [52]. Thus, we tested whether CQ affected pyroptosis in Sham and SCI mice. As revealed in Additional file 1: Fig. S3A–F, IF and WB showed no differences in the pyroptosis-related proteins of Caspase-1, GSDMD-N, NLRP3, NLRP1, ASC, IL-1β and IL-18 between the Sham and Sham+CQ groups. Our results indicated that although CQ treatment inhibited autophagic flux (Fig. 4A–E), it had no significant effect on pyroptosis in non-SCI mice (Additional file 1: Fig. S3A–F). Later, we examined the expression levels of these pyroptosis-related proteins and tested whether CQ attenuated the inhibition of pyroptosis by SS-31 in SCI mice. As shown in Fig. 4F–H, the integrated density of Caspase-1 and NLRP3 in neurons within the SCI+SS-31+CQ group was larger than that within the SCI+SS-31 group based on the IF results. WB demonstrated that these pyroptosis-related proteins were more abundant in the SCI+SS-31+CQ group than in the SCI+SS-31 group (Fig. 4I, J). These investigations indicated that CQ partly reversed the effects of SS-31 in slowing pyroptosis in SCI.
Next, we considered whether CQ mitigated the impact of SS-31 on the functional recovery of SCI mice. We carried out HE/Masson staining, IF, and motor function evaluation among the SCI, SCI+SS-31, and SCI+SS-31+CQ groups 28 days after SCI. The SCI+SS-31+CQ group had lower MAP2 density and fewer Syn-positive synapses than the SCI+SS-31 group, as shown by IF staining (Additional file 1: Fig. S4A–C). Furthermore, the injured area of the spinal cord in the SCI+SS-31+CQ group exhibited a greater glial scar area than that in the SCI+SS-31 group (Additional file 1: Fig. S4D, E). Next, we performed footprint analysis, inclined plane test and BMS score (Additional file 1: Fig. S4F–J). The results showed that the SCI+SS-31 group had clear restoration of hind leg movement with coordinated crawling on Day 28 following damage, but the SCI+SS-31+CQ group continued to drag their hind legs (Additional file 1: Fig. S4F–H). The inclined plane angle and BMS score were considerably lower within the SCI+SS-31+CQ group at 14, 21, and 28 days post-SCI than within the SCI+SS-31 group (Additional file 1: Fig. S4I, J). These investigations showed that SS-31's capacity to increase autophagy could have been responsible for the improved motor function after SCI.
SS-31 attenuates LMP and inhibits cPLA2 phosphorylation after SCI
A previous study has described how LMP and the subsequent release of cathepsin B (CTSB) from lysosomes into the cytosol is a signalling pathway that induces pyroptosis [53]. Additionally, mounting evidence suggests that lysosomal damage impairs autophagic flux in neural diseases [54, 55], such as SCI [6], and leads to the accumulation of neuronal autophagosomes. Given the significant impact of SS-31 on pyroptosis inhibition and autophagic flux restoration in our research, we hypothesized that SS-31 may affect lysosome function following SCI. We prepared lysosome-enriched fractions by subcellular fractionation of spinal cord tissue. Then, we directly identified the presence of lysosomal enzymes using WB and IF. The WB results indicated that the SCI group contained more lysosomal enzymes in the cytosolic fractions (including CTSB, CTSD and CTSL) than the Sham group but lower levels of enzymes in the lysosomal fractions (Fig. 5A–F). IF staining of CTSL and NeuN demonstrated that diffuse CTSL cells were more abundant in SCI mice than in Sham mice (white arrows indicate diffuse CTSL in neurons) (Fig. 5G, H). These results suggested that lysosomal enzymes leaked into the cytosol, demonstrating that LMP occurred in SCI. However, treatment with additional SS-31 in SCI mice changed the distribution of lysosomal enzymes (Fig. 5A–H), which suggested that SS-31 attenuated LMP after SCI.
The findings mentioned above demonstrated that under the pathological conditions of SCI, the ability of the lysosomal membrane to act as a barrier was compromised, enabling lysosomal contents to escape into the cytoplasm. According to a previous study, phosphatides are well known to be critical components of neuronal bilayer membranes [56]. Our group has previously reviewed how the activation of cPLA2/Pla2g4a (phospholipase A2, group IVA [cytosolic, calcium-dependent]) produces phosphatide disintegration and membrane breakdown through the hydrolytic action of neuronal membrane phosphatides, resulting in alterations in membrane functions, such as LMP [57]. Considering SS-31’s effect on protecting the lysosomal membrane from permeabilization in SCI, we assumed that it acted by inhibiting the cPLA2 enzyme. Thus, we examined the levels of cPLA2 and p-cPLA2 in the Sham, SCI and SCI+SS-31 groups. The results showed equivalent expression of cPLA2 in the three groups (Fig. 5I–K). Nevertheless, as shown in Fig. 5I–K p-cPLA2 and the ratio of p-cPLA2/cPLA2 were significantly upregulated by SCI, whereas SS-31 attenuated the enhancement of both upregulations. IF showed similar outcomes: the protein level of p-cPLA2 in the SCI group was higher than that in the Sham group, whereas SS-31 downregulated the p-cPLA2 protein level after SCI (Fig. 5L, M). These results indicated that SS-31 effectively inhibited the activation of cPLA2 in neurons after SCI. Moreover, we performed IF experiments to assess the specific expression of p-cPLA2 in other cell types, including microglia, astrocytes and oligodendrocytes (Additional file 1: Fig. S5A–F). The results showed that the number of p-cPLA2-positive microglia and oligodendrocytes were increased in SCI mice but SS-31 treatment attenuated this upregulation. However, the number of p-cPLA2-positive astrocytes showed no significant differences in the Sham, SCI and SCI+SS-31 groups.
Additionally, the enzymatic activity assay demonstrated that cPLA2 activity increased following SCI and decreased following the administration of SS-31 (Additional file 1: Fig. S6A). Later, we performed subcellular fractionation of spinal cord tissue and prepared lysosome-enriched and cytosolic fractions. ELISA was employed to determine the activity of CTSD and NAGLU in lysosomes. As shown in Additional file 1: Fig. S6B, C, the activity levels of both lysosomal enzymes were lower in the SCI group than in the Sham group. Furthermore, the enzymes were significantly more active within the SCI group's cytosolic fractions than within the Sham group (Additional file 1: Fig. S6D, E). These SCI-induced alterations were significantly attenuated by SS-31 (Additional file 1: Fig. S6B–E), suggesting restoration of lysosomal function. All these results showed that SS-31 inhibited cPLA2 from being phosphorylated and decreased the damage to lysosomal membranes after SCI.
SS-31 attenuates LMP and pyroptosis and enhances autophagy by suppressing the activation of cPLA2 after SCI
To learn more about the function of SS-31-mediated cPLA2 inhibition in attenuating LMP, we employed AAV-Pla2g4a to activate cPLA2 and devised a rescue experiment to compare the following 6 groups: Sham+AAV-Blank, Sham+AAV-Pla2g4a, SCI+AAV-Blank, SCI+AAV-Pla2g4a, SCI+SS-31+AAV-Blank and SCI+SS-31+AAV-Pla2g4a groups. qPCR and WB showed that the application of AAV-Pla2g4a significantly upregulated Pla2g4a mRNA and cPLA2/p-cPLA2 protein expression but did not change the ratio of p-cPLA2 to cPLA2 in the Sham, SCI and SCI+SS-31 groups (Fig. 6A–D). IF staining demonstrated that the protein levels of p-cPLA2 in neurons within the SCI+SS-31+AAV-Pla2g4a group were consistently higher than those within the SCI+SS-31+AAV-Blank group (Fig. 6E, F). All of these findings demonstrated that AAV-Pla2g4a transfection activated cPLA2.
Our team then explored whether LMP is influenced by SS-31's impact on cPLA2. As shown in the WB results in Fig. 6G, H, compared to the SCI+SS-31+AAV-Blank group, the SCI+SS-31+AAV-Pla2g4a group showed a remarkable tendency toward leakage of lysosomal enzymes from lysosomes into the cytoplasm. Based on the IF data (Fig. 6I, J), significantly more diffuse CTSL cells were present in SCI+SS-31+AAV-Pla2g4a mice than in SCI+SS-31+AAV-Blank mice. Recent research has demonstrated that inhibiting cPLA2 not only decreases LMP, but also restores autophagic flux and is connected to reduced neuronal cell damage [6, 19]. In addition, a review focusing on pyroptosis in CNS trauma has proposed that inhibiting cPLA2 may attenuate pyroptosis [53]. Considering the strong ability of SS-31 in the inhibition of cPLA2, we hypothesized that it is responsible for regulating pyroptosis and autophagy. IF staining showed that the integrated density of NLRP3 and Caspase-1 in neurons was significantly lower in the SCI+SS-31+AAV-Blank group than in the SCI+AAV-Blank group, but the opposite results were observed in the SCI+SS-31+AAV-Pla2g4a group (Fig. 7A–C). WB produced a similar outcome: the levels of the pyroptosis-related proteins Caspase-1, GSDMD-N, NLRP3, NLRP1, ASC, IL-1β, and IL-18 were lower in the SCI+SS-31+AAV-Blank group than in the SCI+AAV-Blank group; however, they were elevated in the SCI+SS-31+AAV-Pla2g4a group (Fig. 7G, H). As shown in Fig. 7D–H, the results of IF staining and WB demonstrated that the numbers of LC3 II puncta and the expression of LC3 II were higher in the SCI+SS-31+AAV-Blank group than in the SCI+AAV-Blank group and were significantly lower relative to the SCI+SS-31+AAV-Pla2g4a group. Compared to those in the SCI+AAV-Blank group, the levels of p62 protein according to IF and WB were decreased in the SCI+SS-31+AAV-Blank group and increased in the SCI+SS-31+AAV-Pla2g4a group (Fig. 7D–H). We also carried out experiments on the Sham, Sham+AAV-Blank and Sham+AAV-Pla2g4a groups to test whether overexpressing Pla2g4a affected autophagy, pyroptosis and LMP in non-SCI mice (Additional file 1: Fig. S7A–N). Our results showed that AAV-Pla2g4a injection upregulated the expression of cPLA2 in Sham mice but did not significantly affect autophagy, pyroptosis or LMP (Additional file 1: Fig. S7A–N). In summary, our outcomes demonstrate that SS-31 therapy suppresses cPLA2, which is a crucial approach by which SS-31 slows LMP, inhibits pyroptosis, and promotes autophagy in SCI mice.
Finally, we explored the therapeutic impact of SS-31 following transfection with AAV-Pla2g4a to determine whether cPLA2 is responsible for an improvement in motor function after SCI with SS-31 treatment. As expected, the SCI+SS-31+AAV-Pla2g4a group showed fewer Syn-positive synapses, decreased MAP2 expression in ventral motor neurons (Additional file 1: Fig. S8A, B, D), and a larger area of glial scarring than the SCI+SS-31+AAV-Blank group (Additional file 1: Fig. S8C, E). Footprint analyses demonstrated that the SCI+SS-31+AAV-Pla2g4a mice moved less within their rear legs and exhibited more toe dragging while crawling together than the SCI+SS-31+AAV-Blank mice (Additional file 1: Fig. S8F–H). Furthermore, compared with the SCI+SS-31+AAV-Blank group, the SCI+SS-31+AAV-Pla2g4a mice exhibited a substantially lower BMS score and inclined plane test result at 21 and 28 days following SCI (Additional file 1: Fig. S8I, J). These findings indicate that SS-31's beneficial therapeutic benefits are mediated by enhancement of autophagy, which inhibits pyroptosis as well as LMP by depressing activation of cPLA2.
SS-31 inhibits cPLA2 through the p38-MAPK signalling pathway
Examination of the whole sequence of cPLA2 clearly reveals that this enzyme contains a consensus phosphorylation motif (involving Ser-505) that is a target of the MAPK family, which consists of three major components: ERK, p38, and JNK [58]. In addition, many studies have demonstrated that SS-31 shows great power in inhibiting the activation of the p38 MAPK signalling pathway in a variety of ischaemia‒reperfusion injury disease models [31, 59, 60]. Therefore, we performed further research to learn more about the relationship between SS-31 and the MAPK signalling pathway in SCI. As shown in Fig. 8A, B, p38, ERK1/2, JNK, and cPLA2 were all significantly activated after SCI. The administration of SS-31 reduced the ratio of p-cPLA2/cPLA2 and p-p38/p38, while the ratio of p-ERK1/2/ERK1/2 and p-JNK/JNK did not differ in the SCI+SS-31 group from those in the SCI group. These results suggest that SS-31 inhibits the p38-MAPK pathway rather than the ERK1/2- or JNK-MAPK pathway.
To determine whether the p38-MAPK signalling pathway affected SS-31's impact on cPLA2, we focused on the effects of AA (a known p38 agonist [40]) on the MAPK signalling pathway. The investigations demonstrated that the mice in the SCI+SS-31+AA group had higher p-p38/p38 and p-cPLA2/cPLA2 ratios than those in the SCI+SS-31 group, indicating the efficacy of AA (Fig. 8C, D). As shown in Fig. 8C–E, when compared to the SCI+SS-31 group, Caspase-1, GSDMD-N, NLRP3, ASC, p62, and LC3 II were upregulated in the SCI+SS-31+AA group, demonstrating that AA effectively reversed the SS-31-mediated restoration of autophagic flux and inhibition of pyroptosis in the SCI+SS-31 group. In addition, ELISA demonstrated that the enzymes CTSD and NAGLU were significantly less active within lysosomes in the SCI+SS-31+AA group than in the SCI+SS-31 group. Furthermore, the cytosolic fractions from the SCI+SS-31+AA group had substantially higher activity of both enzymes than those from the SCI+SS-31 group (Additional file 1: Fig. S9A–D). In short, our outcomes show that SS-31 inhibits cPLA2 from becoming active in SCI by blocking the p38-MAPK signalling pathway.