Figures & data
Table 1. The primers for RT-qPCR
Figure 1. Ube2b expression was decreased in CCI rats
CCI rat model was constructed by ligating the left sciatic nerve. Sham-operated rats were served as control. (A) Western blot analysis was performed to assess the expression of Ube2b in spinal cord tissues of rats on 3, 7, and 14 d postoperation. (B) IF staining was utilized for evaluating the level of Ube2b in spinal cord tissues of rats on 3, 7, and 14 d postoperation. N = 6. One-way ANOVA analysis. **p < 0.01 relative to CCI-3 group. CCI: chronic constriction injury; IF: immunofluorescence.
Figure 2. Ube2b elevation alleviated the mechanical hyperalgesia and thermal hyperalgesia in rats
CCI rat model was constructed by ligating the left sciatic nerve. CCI rats and Sham-operated rats were injected with AAV-Ube2b or AAV-NC. The expression of Ube2b in spinal cord tissues of rats was measured via RT-qPCR (A) and Western blot analysis (B). (C) The thermal hyperalgesia and mechanical allodynia of rats were examined via PWTL and PWMT. N = 6. One-way ANOVA analysis. **p < 0.01 relative to Sham + AAV-NC group, ##p < 0.01 relative to CCI+ AAV-NC group. CCI: chronic constriction injury; RT-qPCR: quantitative real-time polymerase chain reaction; PWTL: paw withdrawal thermal latency; PWMT: paw withdrawal mechanic threshold.
Figure 3. Ube2b elevation suppressed chronic sciatic nerve injury
CCI rat model was constructed by ligating the left sciatic nerve. CCI rats and Sham-operated rats were injected with AAV-Ube2b or AAV-NC. (A) H&E staining was used to examine the pathological changes of spinal cord tissues in rats. (B) The level of NeuN in spinal cord tissues of rats was exhibited by IF staining. N = 6. One-way ANOVA analysis. **p < 0.01 relative to Sham + AAV-NC group, ##p < 0.01 relative to CCI+ AAV-NC group. CCI: chronic constriction injury; H&E: Hematoxylin-eosin; IF: immunofluorescence.
Figure 4. Ube2b regulated the expression of DNMT3a and Kcna2
CCI rat model was constructed by ligating the left sciatic nerve. CCI rats and Sham-operated rats were injected with AAV-Ube2b or AAV-NC. The levels of DNMT3a and Kcna2 in spinal cord tissues of rats were displayed by IF staining. N = 6. One-way ANOVA analysis. **p < 0.01 relative to Sham + AAV-NC group, ##p < 0.01 relative to CCI+ AAV-NC group. CCI: chronic constriction injury; IF: immunofluorescence.
Figure 5. Ube2b promoted the level of Kcna2 via regulating DNMT3a
(A) HT22 cells were transfected with Ube2b, NC, shKcna2 or shNC. Western blot analysis was performed to identify the protein levels of Ube2b, DNMT3a, and Kcna2 in the HT22 cells. (B) HT22 cells were transfected with Ube2b, NC, DNMT3a, Vector, shUbe2b, shNC, shDNMT3a, shControl. Western blot analysis was performed to identify the protein levels of Ube2b, DNMT3a, and Kcna2 in the HT22 cells. N = 3. One-way ANOVA analysis. **p < 0.01 relative to NC + Vector group, ##p < 0.01 relative to shNC + shControl group, &&p < 0.01 relative to shUbe2b + shControl group.
Figure 6. Ube2b modulates neuropathic pain via mediating Kcna2
CCI rat model was constructed by ligating the left sciatic nerve, followed by injection of AAV-Ube2b, AAV-shKcna2 or AAV-Ube2b combined with AAV-shKcna2. Sham-operated rats were served as control. (A) IF staining was used for analyzing the levels of Ube2b, DNMT3a and Kcna2 in spinal cord tissues of rats. (B) The thermal hyperalgesia and mechanical allodynia of rats were examined via PWTL and PWMT. N = 6. One-way ANOVA analysis. **p < 0.01 relative to sham group, ##p < 0.01 relative to CCI group, &&p < 0.01 relative to CCI + AAV-Ube2b group. CCI: chronic constriction injury; IF: immunofluorescence; PWTL: paw withdrawal thermal latency; PWMT: paw withdrawal mechanic threshold.
