The expression of LXRβ was decreased in hippocampus of SD mice
To determine whether LXRs signaling was required in SD, the SD model was established using a small platform water environment, in which the mice were deprived of 20 h-sleep a day for 28 d. The expression changes of two subtypes of LXRs in hippocampus were detected after SD. Interestingly, the expression level of LXRβ in SD group was substantially decreased to 37.94% ± 5.23% of Ctrl (p < 0.001, SD group vs. Ctrl; Fig. 1b), while the expression level of LXRα in hippocampus was not altered after SD (p > 0.05, SD group vs. Ctrl; Fig. 1b). These data suggested that SD elicited the reduction of LXRβ expression in hippocampus and activation of LXRβ with an agonist might alleviate SD-induced symptoms.
LXRs agonist GW3965 alleviated SD-induced cognitive impairment
Cognitive impairment is one of the most important symptoms induced by SD . In this study, two classic behavioral methods, MWM and NOR were used to examine the cognitive changes of mice from each group. The experimental process was shown in Fig. 1a. As expected, SD mice spent more time to find the platform in MWM test, which was manifested as an increase in escape latency of 39.01± 2.61 s at d 4, compared with Ctrl group of 10.89 ± 2.24 s (p < 0.001 at d 4, SD group vs. Ctrl; Fig. 1c). The swimming distance in SD group was 14.82 ± 7.47 m at d 3 and 12.68 ± 1.26 m at d 4, while Ctrl group was 8.43 ± 0.98 m at d 3 and 3.70 ± 0.90 m at d 4 (p < 0.01 at d 3, p < 0.001 at d 4, SD group vs. Ctrl; Fig. 1d). This result indicated that SD mice swam longer distances than Ctrl group before finding the platform. However, the escape latency and swimming distance in GW-treated group were significantly decreased to 14.90 ± 1.79 s at d 4 (p < 0.001, SD + GW group vs. SD group; Fig. 1c) and 5.69 ± 0.58 m (p < 0.01, SD + GW group vs. SD group; Fig. 1d). This result indicated that GW treatment ameliorated SD-induced learning impairment. During the probe trials, SD-induced impaired memory, which was manifested as a decrease in time spent in the target quadrant and platform crossings compared with Ctrl group (p < 0.01, SD group vs. Ctrl; Fig. 1e; p < 0.001, SD group vs. Ctrl; Fig. 1f). However, the impaired memory was improved after GW administration (p < 0.05, SD + GW group vs. SD group; Fig. 1e, f). In NOR test, SD decreased the time spent on exploring the novel object. The discrimination index in SD group was 49.28% ± 2.62% compared with Ctrl group of 69.05% ± 2.66% (p < 0.001, SD group vs. Ctrl; Fig. 1g), indicating that SD impaired the memory of mice. As expected, GW treatment alleviated this decrease to 61.23% ± 1.80% (p < 0.01, SD + GW group vs. SD group; Fig. 1g). Finally, to exclude the interference of locomotor activity, the locomotor abilities of different groups were analyzed and there were no differences among the three groups (p > 0.05; Fig. 1h). Consistent with previous studies, SD-induced cognitive impairment was observed in our study. The result also proved that activating LXRs by GW alleviated cognitive impairment after SD.
Knockdown of LXRβ by shRNA in hippocampus led to cognitive impairment
It had been proved that SD led to a significant decrease of LXRβ expression in hippocampus. This result suggested that hippocampal LXRβ defects might be associated with cognitive impairment. Given the lack of selective antagonists for LXRβ isoform, we used shLXRβ stereotactic injection into the hippocampus to knock down endogenous LXRβ. Western blot data showed that the level of LXRβ was decreased to 33.61% ± 2.28% of shNC (p < 0.001, shLXRβ group vs. shNC; Fig. 2a) after shLXRβ transfection. WMZ result indicated that the mice in shLXRβ group spent more time and traveled longer distance to find the platform (p < 0.001 at d 4, shLXRβ group vs. shNC; Fig. 2b, c). Compared with the shNC group, the time spent in the target quadrant and platform crossings of shLXRβ group were also decreased (p < 0.05, shLXRβ group vs. shNC; Fig. 2d, p < 0.01, shLXRβ group vs. shNC; Fig. 2e). In NOR test, shLXRβ group showed a decrease in discrimination index (p < 0.01, shLXRβ group vs. shNC; Fig. 2f). These results indicated that reduced hippocampal LXRβ expression was associated with cognitive impairment. However, when LXRα expression was decreased to 43.38% ± 3.78% of shNC (p < 0.001, shLXRα group vs. shNC; Fig. 2a) after shLXRα infection in hippocampus, the mice showed no cognitive impairment compared with shNC mice (p > 0.05, shLXRα group vs. shNC; Fig. 2b-f). Furthermore, combined shLXRα + shLXRβ infection could not lead to further reduction of LXRα and LXRβ expression and more severe cognitive impairment (Fig. 2).
GW3965 improved SD-induced cognitive impairment by activating LXRβ isoform
Based on the previous results, it seemed that LXRβ isoform played a much more important role in cognition than LXRα isoform. Since GW was a non-selective agonist for both LXRα and LXRβ subtypes, shRNA-mediated knockdown of LXRα and LXRβ in hippocampus was also used to verify the subtype of GW-mediated cognitive improvement. The SD model was set up 7 d after shRNA infection, and mice were treated as before (Fig. 3a). After shLXRα infection, GW could still improve SD-induced learning disabilities (p < 0.001 at d 4 shLXRα + SD + GW group vs. shLXRα + SD group; Fig. 3b, c) and memory impairment (p < 0.05 shLXRα + SD + GW group vs. shLXRα + SD group; Fig. 3h and p < 0.01 shLXRα + SD + GW group vs. shLXRα + SD group; Fig. 3i). This result indicated that knockdown of hippocampal LXRα expression did not affect GW-mediated cognitive improvement in SD mice. However, the knockdown of hippocampal LXRβ prevented GW-mediated cognition improvement (p > 0.05, shLXRβ + SD + GW group vs. shLXRβ + SD group; Fig. 3d, e, h, i). Furthermore, when the expression levels of LXRα and LXRβ were both down-regulated, GW also failed to exert its effects (p > 0.05, shLXRα + β + SD + GW group vs. shLXRα + β + SD group; Fig. 3f-i). These results suggested that GW relieved SD-induced cognitive impairment by activating LXRβ isoform.
GW relieved SD-induced microglia activation, followed by suppressing HMGB1/TLR4/NF-κB p65 pathway in hippocampus
It had been shown that GW improved SD-induced cognitive impairment through LXRβ, but the mechanisms involved were still unclear. Evidence showed that knockout of LXRβ led to microglia activation, suggesting LXRβ was responsible for neuroinflammation (Colonna & Butovsky, 2017; Dai, Tan, Wu, Warner, & Gustafsson, 2012). In this study, we found that hippocampal microglia were activated with a decreased LXRβ expression (Extended Data Fig. 1), suggesting that the status of microglia might be related to the expression level of LXRβ upon SD injury. Besides, the presence of LXRβ in the microglia was also observed (Extended Data Fig. 2). To verify whether GW could inhibit microglia activation after SD, the status of microglia in SD + GW group was tested. As expected, activated microglia in SD mice were significantly reduced after GW administration (Fig. 4a). Western blot results also showed that SD robustly enhanced the hippocampal expression level of Iba-1 to 235.30% ± 14.22% of Ctrl (p < 0.001, SD group vs. Ctrl; Fig. 4b, c), while GW treatment decreased this level to 121.30% ± 5.67% of Ctrl (p < 0.001, SD + GW group vs. SD group; Fig. 4b, c). These results confirmed that GW could mitigate microglia activation induced by SD in hippocampus.
HMGB1 is a typical damage-associated molecule involved in various neuroinflammatory and neurodegenerative diseases, and the activation of HMGB1/TLR4/NF-κB p65 pathway is highly associated with microglial activation and leads to elevated level of inflammatory factors. In this study, we found that the hippocampal expression of HMGB1 and TLR4 was increased to 151.00% ± 7.50% and 205.30% ± 7.70% of Ctrl after SD (p < 0.001, SD group vs. Ctrl; Fig. 5a, b, d, e). Besides, SD also increased nuclear level of NF-κB p65 and decreased cytoplasmic NF-κB p65 expression correspondingly in hippocampus (p < 0.001, SD group vs. Ctrl; Fig. 5g-i), leading to elevated TNF-α and IL-1β (p < 0.001, SD group vs. Ctrl; Fig. 5c, f). However, GW treatment could significantly reduce the protein levels of HMGB1, TLR4, nuclear NF-κB p65, TNF-α and IL-1β (SD + GW group vs. SD group; Fig. 5a-i), suggesting that GW could ameliorate neuroinflammation in hippocampus by inhibiting the HMGB1 pathway.
GW suppressed HMGB1/TLR4/NF-κB p65 pathway in activated microglia by activating LXRβ
The inhibitory effects of GW on HMGB1 pathway in activated microglia were further confirmed in cultured murine N9 microglia in vitro. GW pre-treatment dose-dependently decreased the induction of Iba-1 and CD68, a marker of activated microglia, induced by LPS/ATP stimulation shown in extended data Fig. 3. On account of no obvious differences between 10 μM and 100 μM group, 10 μM of GW was used in the following trials.
To verify whether the inhibitory effect of GW was through LXRβ, shLXRβ was further applied in cultured N9 microglia. Western blot data showed that the expression of LXRβ was successfully down-regulated to 35.92% ± 4.45% of shNC after shLXRβ infection for 3 d (p < 0.001, shLXRβ group vs. shNC; Fig. 6a). LPS/ATP stimulation robustly enhanced the expression levels of HMGB1, TLR4, nuclear NF-κB p65, TNF-α and IL-1β, which were relieved by the pre-treatment of GW as shown in Fig. 6. However, the proteins in HMGB1 pathway were increased in shLXRβ + LPS/ATP + GW group compared with shNC + LPS/ATP + GW group, and showed little difference compared with shLXRβ + LPS/ATP group (Fig. 6b-h). The results indicated that GW inhibited HMGB1/TLR4/NF-κB p65 pathway in activated microglia due to the presence of LXRβ.
HMGB1, TNF-α and IL-1β were released from cells to exert the pro-inflammatory roles, then the content changes in the supernatant were also detected by ELISA assay. The ELISA data, showed in extended data figure 4, exhibited similar tendency to that of Western blot results as showed in Fig. 6. The expression of HMGB1, TNF-α and IL-1β in the supernatant showed little difference between shLXRβ + LPS/ATP + GW group and shLXRβ + LPS/ATP group, which meant GW inhibited inflammatory cytokines released from N9 microglia by activating LXRβ.
Inhibition of HMGB1 facilitated the anti-inflammatory effect of GW upon LPS/ATP-induced inflammatory responses in murine N9 microglia
To further confirm that HMGB1 pathway exerted an important role in GW-mediated anti-inflammatory effects, a direct HMGB1 inhibitor GLY was used . The results showed that GLY (50 μM) significantly inhibited nuclear translocation of NF-κB p65 upon LPS/ATP stimulation (LPS/ATP + GLY group vs. LPS/ATP group; Fig. 7a, b), followed by the expression reductions of TNF-α and IL-1β (LPS/ATP + GLY group vs. LPS/ATP group; Fig. 7a, c) and the release reduction into the supernatants (LPS/ATP + GLY group vs. LPS/ATP group; Fig. 7d). Furthermore, GLY administration enhanced GW-mediated the reduction of nuclear NF-κB p65 upon LPS/ATP injury, as well as the release of TNF-α and IL-1β (LPS/ATP + GW + GLY group vs. LPS/ATP + GW group; Fig. 7). This result indicated that GLY could promote the anti-inflammatory effects of GW in activated microglia.
Inhibition of HMGB1 pathway effectively alleviated SD-induced cognitive impairment and enhanced GW-mediated cognitive improvement
Finally, the role of HMGB1 pathway in SD-induced cognitive impairment was tested. GLY (20 mg/kg) and/or GW (10 mg/kg) were injected intraperitoneally (i.p.) starting from d 14 after SD, for 14 d consecutively (Fig. 8a). Behavior test showed that treatment with GLY alone for 14 d effectively alleviated SD-induced learning and memory deficit (SD + GLY group vs. SD group; Fig. 8b-f). Furthermore, GW and GLY had synergistic effects in improving cognitive impairment, which was manifested as a tendency to increase learning and memory functions compared with using GW or GLY alone (SD + GW + GLY group vs. SD +GW or SD + GLY group; Fig. 8b-f). Collectively, all the results confirmed that HMGB1 pathway played an important role in cognitive deficit, and the inhibition of HMGB1 enhanced GW-mediated cognitive improvement after SD.