Piperine promotes functional recovery
The BMS score were used to assess locomotor function recovery in the Sham, Vehicle, Piperine and Piperine + 3MA groups pre-SCI and every 2 days post-SCI. After surgery, both BMS score were significantly higher in the Sham group than in the Vehicle group at different time points; after piperine treatment, the SCI group had higher BMS score than the SCI group. However, the changes were attenuated by 3-MA (Figure 1A). In the inclined plane test, we then tested the inclined plane angle, and in the footprint analysis test, we measured the rotation angle, base of support and stride length in the 4 four groups. The inclined plane angle and stride length results showed that the mice in the Vehicle group had lower scores than those in the Sham group, while piperine restored the scores, and 3-MA reversed the effect of piperine (Figure 1B, E). Moreover, the rotation angle and base of support in the Vehicle group were higher than those in the Sham group. In contrast, piperine decreased the data above, while 3-MA inhibited the effects of piperine (Figure 1C, D).
Piperine enhances autophagy after SCI
To evaluate the level of autophagy at the spinal cord lesion after SCI, we measured the levels of autophagosomal markers (Beclin1, ATG5, VPS34 and LC3II), autolysosome-related markers (CTSD and ATP6V1B2) and a substrate protein (p62). The immunofluorescence results showed that the percentage of LC3II-positive neurons was higher in the SCI group than in the Sham group. Furthermore, piperine treatment further increased the percentage of LC3II-positive neurons in SCI mice (Figure 2A, B). Similarly, the percentage of p62-positive neurons increased substantially after SCI, while the percentage of p62-positive neurons was lower in the Piperine group than in the SCI group (Figure 2C, D). The Western blotting results showed the expression levels of Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II and p62 (Figure 2E). The optical density (OD) values for Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II and p62 were higher in the Vehicle group than in the Sham group (Figure 2F–L). In addition, the changes in the OD values of these markers (except p62) were enhanced in the Piperine group (Figure 2F–L).
Piperine attenuates oxidative stress after SCI
Considering that SCI might result in oxidative stress, we next determined the effects of piperine on SOD, MDA, eNOS and HO-1 levels in the spinal cord. The level of SOD in the tissue was measured as an index of tissue antioxidant activity, and the level of MDA was measured as an index of the lipid level in the tissue. The activity of SOD decreased after SCI, while piperine attenuated the change (Figure 3A). Compared with the Sham group, the SCI group showed a significant decrease in the levels of SOD, eNOS and HO-1. Interestingly, piperine significantly attenuated the abnormally decreased levels of these markers (Figure 3 C, D, E, F). In addition, compared with the Sham group, the SCI group showed an abnormal increase in the level of MDA. However, piperine significantly attenuated the abnormally increased levels of MDA after SCI (Figure 3B).
Piperine attenuates pyroptosis after SCI
To assess the status of pyroptosis, we evaluated the pyroptosis biomarkers NLRP3, GSDMD, C-CASP1, IL-18 and IL-1β to determine the pyroptosis level in the Sham, SCI + Vehicle and SCI + Piperine groups. As shown in Figure 4A, immunofluorescence staining was performed to show the expression of C-CASP1 in neurons at the lesion (C-CASP1 is labeled in green, the neurons are labeled in red, and the nuclei are labeled in blue). Our results showed that the fluorescence intensity of C-CASP1 was much brighter and the number of C-CASP-1-positive cells was much greater in the SCI group than in the Sham group, but piperine significantly decreased the fluorescence intensity of C-CASP1 and diminished the number of C-CASP-1 positive cells (Figure 4A, B). As shown in Figure 4B, the Western blotting results revealed the expression levels of NLRP3, GSDMD, and C-CASP1 (Figure 4C). The OD values of NLRP3, GSDMD, and C-CASP1 were higher in the Vehicle group than in the Sham group, while piperine attenuated these changes (Figure 4D–F). The levels of IL-1β and IL-18 were detected by ELISA, and the results showed that both IL-1β and IL-18 levels in the Vehicle group were increased compared with those in the Sham group. Meanwhile, piperine significantly reversed the alterations in IL-1β and IL-18 levels in the mice after SCI (Figure 4G, H).
Piperine suppresses inflammation after SCI
Three days after SCI, we used ELISA to evaluate the protein concentrations of proinflammatory cytokines, including IL-1β, IL-6, TNF-α and IFN-γ, in the spinal cord of the three groups. Owing to the profound impact of the injured spinal cord, the SCI group had significantly increased protein concentrations of proinflammatory cytokines compared to the Sham group, while the administration of piperine significantly reversed the effects of SCI (Figure 5A–D). Because incorrect interpretation of inflammation could result when only the protein levels of these proinflammatory cytokines are measured, we used PCR to detect the mRNA levels of these proinflammatory cytokine proteins. Compared with the Sham group, the SCI group exhibited increased IL-1β, IL-6, TNF-α and IFN-γ mRNA levels, indicating that increased transcription of proinflammatory cytokines may be directly related to SCI. Compared with the SCI group, the Piperine group exhibited significantly lower relative cytokine mRNA levels, suggesting that decreased transcription of these cytokines may be related to treatment with piperine (Figure 5H–K). We also detected the expression levels of proinflammatory factors, such as iNos and COX-2. Western blotting revealed that the expression levels of Cox-2 and iNos were increased in the SCI group compared to the Sham group; however, they were inhibited by piperine treatment in the SCI+Piperine group compared with the SCI group (Figure 5E-G).
Inhibition of autophagy reverses the effect of piperine on oxidative stress, pyroptosis, inflammation and functional recovery after SCI
After autophagy inhibition, the levels of autophagy, oxidative stress, pyroptosis and inflammation in SCI were evaluated by Western blotting. The expression levels of Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II, and p62 were detected by Western blotting (Figure 6A). The results revealed that the OD values of Beclin1, ATG5, VPS34, CTSD, ATP6V1B2 and LC3II were lower in the SCI+Piperine+3MA group than in the SCI + Piperine group, while a higher OD value of p62 was observed in the SCI + Piperine group (Figure 6B). We also detected the expression levels of Cox-2, iNOS, NLRP3, GSDMD, C-CASP1, SOD1, eNOS and HO-1 by Western blotting (Figure 6 C-E). The OD values of Cox-2, iNOS, NLRP3, GSDMD, C-CASP1, SOD1, eNOS and HO-1 were higher in the SCI+Piperine+3MA group than in the SCI+ Piperine group (Figure 6F–H). We then measured the levels of IL-1β and IL-18 by ELISA, and the results revealed that both IL-1β and IL-18 levels in the SCI+Piperine+3MA group were increased compared with those in the SCI+ Piperine group (Figure 6I, J).