DJ-1 Interference Aggravated Injury after Cerebral I/R Injury
HE and Nissl staining were conducted to assess morphological changes in the ischemic lesions 24 h after reperfusion (Fig. 1A,C). HE staining demonstrated that neurons in the MCAO and NC groups adopted a more disorderly arrangement than in the sham group and exhibited loosened cytoplasm and karyopyknosis. However, transfection with DJ-1 siRNA increased the level of diffuse vacuolization and edema in the interstitial area and decreased the number of intact neurons. Results from Nissl staining were similar to those from HE staining. In contrast, a large number of atrophic neurons that showed damaged nuclei and shrunken cytoplasm were observed in the MCAO and NC groups. Most neurons showed a loss of Nissl bodies in the DJ-1interference group than in the MCAO groups. Cerebral infarct volume and neurological deficit scores were used to further confirm the neuroprotective role of DJ-1. As shown in Fig. 1B, D, no infarction was observed in the sham group. The infarct volume was significantly increased in the DJ-1 siRNA group compared with the MCAO group. As shown in Fig. 1E, compared with the MCAO group, neurological deficit scores increased after rats were infected with DJ-1siRNA.
The CCK-8 assay was conducted to evaluate the roles of DJ-1 in astrocyte viability after exposure to OGD/R. Cells in the OGD/R group and NC group were less viable than those in the control group, and viability was further decreased in the DJ-1 siRNA group （Fig. 1F）. These results suggest that DJ-1 interference can aggravate cerebral I/R injury.
DJ-1 Interference Increased the Expression of TNF-α, IL-1β, and IL-6 after Cerebral I/R Injury
To determine whether DJ-1 is involved in inflammation, we analyzed the expression of inflammatory cytokines after interfering with DJ-1. Western blot analysis and ELISA were conducted to detect the expression levels of TNF-α, IL-1β, and IL-6. In vivo TNF-α, IL-1β, and IL-6 expression in MCAO group was increased compared with that in the sham group, and the expression of these cytokines was further increased in the DJ-1 siRNA group (Fig. 2A, C, E-G). Similar results were observed in vitro (Fig. 2B, D, H-J).
DJ-1 Regulated NLRX1, TRAF6, SHP-1 after Cerebral I/R Injury
NLRX1, a recently characterized member of the NOD-like family, is widely expressed in the mitochondria. We assessed mitochondrial NLRX1 expression using the mitochondria-specific marker HSP60. As shown in Fig. 3E, F, NLRX1 was expressed in the mitochondria. NLRX1, which is known to negatively regulate inflammation, also negatively regulated TLR-induced NF-kB signaling by dissociating from TRAF6 . Due to DJ-1 is also an anti-inflammatory regulator, we investigated whether DJ-1 regulates the expression of NLRX1, TRAF6 after cerebral I/R injury. The expression of NLRX1 was significantly upregulated following cerebral I/R injury compared to that in control samples. However, changes in DJ-1 had no effect on levels of NLRX1 in vivo and in vitro (Fig. 3A-D). After MCAO, the expression of TRAF6 increased compared with that in the sham group and was further increased in the DJ-1 siRNA group (Fig. 3A, C). Similar results were observed in vitro (Fig. 3B, D). Similarly, SHP-1 (Src-homology 2 domain-containing phosphatase-1) acts as a critical negative regulator of inflammatory response , we thus assessed the expression of SHP-1. After MCAO, the expression of SHP-1 was higher than that in the sham group, and after DJ-1 knockdown, the expression of SHP-1 was lower than that in the MCAO group (Fig. 3A, C). Similar results were observed in vitro (Fig. 3B, D).
DJ-1 Regulated the Interaction of TRAF6 with NLRX1 and SHP-1 after Cerebral I/R Injury
NLRX1 inhibits TLR-mediated NF-κB signaling by dissociating from TRAF6 , we thus examined whether DJ-1 regulates the interaction between NLRX1 and TRAF6 after cerebral I/R injury. NLRX1 interacted with TRAF6 in the sham group; however, NLRX1 disassociated from TRAF6 after cerebral I/R injury. Interestingly, DJ-1 interference promoted the interaction of NLRX1 with TRAF6 (Fig. 4A, C). These results indicate that NLRX1 may associate with TRAF6 in resting cells. After cerebral I/R injury, NLRX1 may disassociate from TRAF6 and thus inhibit inflammation. However, DJ-1 interference prevented the dissociation of NLRX and TRAF6 and thus enhanced inflammation. Since TRAF6 plays a prominent role in inflammation by NF-κB activation, and a previous study demonstrated the association between SHP-1 and TRAF6 in RANKL-stimulated BMMs . Therefore, we hypothesized that TRAF6 interacts with SHP-1. We assessed the interaction between SHP-1 and TRAF6 after astroglial DJ-1 interference in cerebral I/R injury. SHP-1 was associated with TRAF6 in the MCAO group but not in the sham group. These interactions were inhibited significantly after treatment with DJ-1 siRNA (Fig. 4B, D).
As shown in Fig. 4A, C, DJ-1 interference promoted the interaction of NLRX1 with TRAF6. These results suggest that DJ-1 facilitate the dissociation of TRAF6 from NLRX1 and may be related to the interaction of TRAF6 with SHP-1.
DJ-1 Inhibited the Expression of TNF-α, IL-1β, and IL-6 after Cerebral I/R Injury via SHP-1
SHP-1 has a strong negative regulator of inflammation effects, and SHP-1-deficient mouse brains show enhanced inflammation . To determine whether SHP-1 is involved in inflammation, TPI-1 was used to inhibit the expression of SHP-1. As shown in Fig. 6E-H, inhibition of SHP-1 increased the expression of TNF-α, IL-1β, and IL-6 compared with that in the DMSO group in vivo and vitro. However, inhibition of SHP-1 had no effect on the level of DJ-1. Interestingly, DJ-1 can regulate SHP-1, we assessed whether DJ-1 inhibits cytokine levels after stroke via SHP-1. Virus and TPI-1 were used to overexpress the DJ-1 and inhibit the SHP-1. Overexpression of DJ-1 reduced the expression of TNF-α, IL-1β, and IL-6 compared with that in the MCAO group (Fig. 5A, C, I, K, M). After treatment with SHP-1 inhibitor, the expressions of TNF-α, IL-1β, and IL-6 were increased. Similar results were obtained in vitro (Fig. 5B, D, J, L, N). Thus, DJ-1 exerts anti-inflammatory effects in astrocytes in cerebral I/R injury via SHP-1.
DJ-1 Regulated the Disassociation of NLRX1 and TRAF6 after Cerebral I/R Injury via SHP-1
To determine whether the DJ-1-induced dissociation of NLRX1 and TRAF6 occurs via SHP-1, Western blotting was conducted to assess the expression of NLRX1, TRAF6, and SHP-1, and immunoprecipitation was used to detect the interactions between NLRX1 with TRAF6 and TRAF6 with SHP-1. As shown in Fig. 6A-D, G- J, Overexpression of DJ-1 increased SHP-1 levels, and treatment with TPI-1 reduced SHP-1 levels in vivo and vitro. However, changes in DJ-1 or SHP-1 levels had no effect on levels of NLRX1 in vivo and in vitro. Figure 6A, G shows that overexpressing DJ-1 reduced TRAF6 expression compared with that in the MCAO group, and TRAF6 expression was increased after treatment with a SHP-1 inhibitor. Similarly, TRAF6 levels after only treatment with a SHP-1 inhibitor were measured. Figure 6C, I shows that inhibition of SHP-1 increased TRAF6 levels. Similar results were obtained in vitro (Fig. 6B, D, H, J). The overexpression of DJ-1 facilitated the interaction of SHP-1 with TRAF6, which was inhibited after treatment with SHP-1 inhibitor. However, NLRX1 disassociated from TRAF6 with DJ-1 overexpression, and SHP-1 inhibition promoted the interaction of NLRX1 with TRAF6 (Fig. 6E, F). Thus, SHP-1 plays a vital role in the disassociation of NLRX1 and TRAF6, which is regulated by DJ-1.