1. Expression and distribution of PDCD4 in the spinal cord of CCI mice
We first examined the expression of PDCD4 in the spinal cord of CCI mice at different time points after surgery. Western blot results revealed a significant increase in PDCD4 expression at 3, 7,14 and 21 days post-CCI (Fig.1 A). To determine the cellular localization of PDCD4 in the spinal cord of CCI mice, we performed immunofluorescence co-staining of PDCD4 with neuronal marker NeuN, astrocyte marker GFAP, and microglia marker IBA1. The immunofluorescence results demonstrated predominant expression of PDCD4 in spinal cord neurons and microglia, with minimal expression observed in astrocytes (Fig.1 B).
2. The impact of intrathecal adenovirus-mediated PDCD4 inhibition on behavioral outcomes in CCI mice.
Previous investigations have revealed an upregulation of PDCD4 expression in the spinal cord of CCI mice. In this part, we employed an innovative tool, AAV-shPDCD4, to suppress the expression of PDCD4. The construction of AAV-encoded shPDCD4-EGFP or control EGFP was shown in Fig. 2A. Immunofluorescence staining results showed the expression of GFP in the spinal cord dorsal horn (Fig.2 B). Then, the expression of PDCD4 following AAV-shPDCD4-EGFP and AAV-EGFP delivery has been examined in the spinal cord (Fig.2 C). Results showed that PDCD4 protein was significantly suppressed by AAV-encoded shPDCD4 EGFP in the spinal cord compared with the AAV-control EGFP group. There was no significant difference in pain behaviors among the groups before or after shPDCD4-EGFP or control injection. Timecourse of pain behaviors showed that PDCD4 inhibition had a significant inhibitory effect on the PWL (Fig.3 B) and PWT (Fig.3 C) evaluation revealed that mechanical allodynia and thermal allodynia were attenuated in AAV- shPDCD4-EGFP treated mice at all time points after CCI compared with AAV-control-treated mice. However, there was no significant change in pain behaviors in the AAV-control group during the whole experiment. These results revealed that inhibition of PDCD4 in the spinal cord is responsible for pain relief induced by CCI.
3. The influence of intrathecal AAV-shPDCD4 injection on spinal cord autophagy in CCI mice
Enhanced autophagy has been shown to alleviate neuropathic pain in mice[27]. PDCD4 is an important molecule in regulating autophagy, and its regulation is involved in various physiological and pathological processes, such as gastric cancer, endometriosis, and pancreatic cancer[28-30]. Our findings thus far suggested that PDCD4 regulated neuropathic pain in CCI mice. To uncover the underlying mechanisms, here we investigated the relationship between PDCD4 and autophagy in neuropathic pain regulation. As shown in Fig.4, western blot results indicated that intrathecal AAV-shPDCD4 injection significantly enhanced the expression of Beclin1and LC3B, while reducing the expression of p62. These results suggested that PDCD4 may participate in neuropathic pain regulation through the modulation of autophagy.
4. The impact of intrathecal AAV-shPDCD4 injection on MAPK pathway activation
Studies have demonstrated that PDCD4 is a crucial regulator of inflammatory responses, and the MAPK pathway plays a pivotal role in regulating neuroinflammation[31]. Previous researches have indicated that PDCD4 modulated the MAPK pathway in processes such as tumor development and fulminant hepatic failure[32, 33]. In this study, we examined the effects of PDCD4 inhibition on MAPK pathway activation. Immunofluorescence results indicated that intrathecal AAV-shPDCD4 injection suppressed the expression of p-ERK, p-JNK, and p-p38MAPK in the spinal cord after CCI (Fig.5). These findings suggested that PDCD4 may regulate neuropathic pain through the modulation of the MAPK pathway activation.
5. The impact of intrathecal AAV-shPDCD4 injection on glial activation and neuroinflammation
PDCD4 plays an important role in regulating neuroinflammation, studies have shown that PDCD4 was upregulated in the cerebral cortex of LPS induced neuroinflammation, and inhibition of PDCD4 inhibits microglial activation through the MAPK pathway[17]. Glial activation is one of the important manifestations of neuroinflammation[34]. Given the important regulatory role of PDCD4 in neuroinflammation, we investigated the effect of PDCD4 inhibition on glial activation. As shown in Figure 6, the expression of activated markers GFAP and IBA1 in astrocytes and microglia is inhibited by adeno-associated virus inhibition of PDCD4 within the subarachnoid space. At the same time, we also tested the expression of inflammatory factors, ELISA results further demonstrated a reduction in the expression of inflammatory factors IL-6, IL-1β, and TNF-α following AAV-mediated PDCD4 inhibition (Fig.5 B-D). These findings suggested that PDCD4 may regulate neuropathic pain through the modulation of the activation of astrocytes and microglia and neuroinflammation.
6. Autophagy activation was required for PDCD4 inhibition induced analgesia
Above studies have confirmed that inhibiting spinal cord PDCD4 alleviated pain hypersensitivity in CCI mice, possibly through activation of autophagy. To further clarify the regulatory relationship between PDCD4 and autophagy, we confirmed it through the following experiments. Two weeks following direct injection of AAV-shPDCD4, the mice were established neuropathic pain model by CCI. To assess the contribution of autophagy in the spinal cord of CCI mice, an autophagy inhibitor 3-MA (i.t., 10μL, 5 μg/d/mouse) or vehicle (10 μL) was given within the sheath once daily from day 7 to day 9 after CCI (Fig. 7A). As shown in Fig. 7 B-C, treatment with 3-MA markedly reversed the analgesic effect of PDCD4 inhibition, the change of p62, LC3B and Becline1 induced by PDCD4 inhibition in CCI mice were also reversed by 3-MA (Fig.7 D-F). Moreover, the reduction of inflammatory cytokines was reversed by 3-MA as well (Fig.7 G-I). These results going further suggested PDCD4 may regulated neuropathic pain via autophagy.