脊髓损伤
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Figure 1｜Lithium promotes recovery of motor function and tissue repair in rats with spinal cord injury (SCI).
To investigate whether lithium can improve motor function in rats with SCI, we determined BBB scores and performed inclined plate tests at 1, 3, 5, 7, 14, and 28 days after surgery in SCI rats. As shown in Figure 1, while the rats in the sham group had no dyskinesia, those in the other two groups had severe dyskinesia that gradually resolved over the duration of the experiment. In addition, the BBB scores in the lithium group were significantly higher than those in the SCI group from the 5th day after surgery (P < 0.05; Figure 1A). Similar results were obtained with the inclined plated test (Figure 1B). Taken together, these results show that lithium can improve mobility in SCI rats.
Figure 2｜Lithium suppresses activation of inflammatory cytokines in the spinal cord of rats with SCI.
The levels of IL-6, IL-1β, and TNF-α expression in rat spinal cord tissue after SCI were detected by enzyme-linked immunosorbent assay to evaluate inflammation (Figure 2A–C). TNF-α was expressed at significantly higher levels in the SCI group than in the sham group. Lithium administration significantly reduced TNF-α expression compared with the SCI group (P < 0.05). The levels of IL-6 and IL-1β exhibited similar trends, as they were significantly lower in the lithium group compared with the SCI group (Figure 2A–C). As shown in Figure 2D, immunohistochemical staining showed that the number of IL-1β-positive cells in the SCI group was significantly higher than that in the sham group (P < 0.05). After lithium treatment, the positive staining in the spinal cord tissue was significantly weaker than that in the SCI group (P < 0.05; Figure 2E). Consistent with this, western blotting revealed that TNF-α, IL-6, and IL-1β protein expression was significantly higher in the spinal cord tissue of SCI rats than in the sham group (P < 0.05; Figure 2F and G). Treatment with lithium reduced the expression levels of these inflammatory factors compared with the SCI group (P < 0.05). These results strongly suggest that lithium treatment mitigates SCI by activating anti-inflammatory pathways.
Figure 3｜Lithium reduces oxidative damage in the spinal cord of rats with SCI.
CAT, GSH-Px, LPO, SOD, MDA, and T-AOC are markers of oxidative changes that occur with injury. In the SCI group, CAT, GSH-Px, SOD, and T-AOC levels were decreased, and LPO and MDA levels were significantly increased, compared with the sham group (P < 0.05). In the lithium group, LPO and MDA levels were decreased, and CAT, GSH-Px, SOD, and T-AOC levels were significantly increased compared with the SCI group (all P < 0.05; Figure 3A–F). Additionally, there was a marked increase in DHE fluorescence intensity (indicating ROS) after SCI. Lithium treatment resulted in significantly decreased mean ROS fluorescence intensity compared with the sham group (P < 0.05; Figure 3G). These results indicate that, in addition to promoting the expression of anti-inflammatory factors, lithium also protects the spinal cord against injury through an anti-oxidant mechanism.
Figure 4｜Lithium inhibits pyroptosis in the spinal cord of rats with SCI.
Pyroptosis plays a special role in the pathogenesis of SCI. Increased Caspase-1 expression and activation are crucial to the progress of pyroptosis after induction of SCI (Dai et al., 2019). Therefore, we performed immunohistochemical staining of spinal cord tissue for Caspase-1 7 days after SCI. Only a small number of Caspase-1-positive cells were observed in the sham group, while more Caspase-1-positive were observed in the SCI group. The number of Caspase-1-positive cells was significantly lower in the lithium group than in the SCI group (P < 0.05; Figure 4A and B), suggesting that lithium significantly suppressed the activation of Caspase-1 in SCI. Western blotting showed that NLRP3, ASC, Pro-caspase-1, Caspase-1, GSDMD, and IL-18 were expressed at significantly higher levels in the SCI group than in the sham group, while treatment with lithium significantly decreased expression of these proteins (P < 0.05; Figure 4C and D). Taken together, these results suggest that lithium inhibited pyroptosis after SCI in rats.
Figure 5｜Lithium up-regulates the Nrf2/HO-1 signaling pathway in the spinal cord of rats with SCI.
As shown in Figure 5A and B, compared with the sham group, the protein expression levels of nuclear Nrf2 and cytoplasmic HO-1 were slightly increased in the SCI group. The slight increase in Nrf2 expression may have been associated with the stress response to a severe stimulus. In agreement with the western blot results, immunofluorescence staining showed an increasing trend in Nrf2 and HO-1 expression after SCI. Nuclear Nrf2 and cytoplasmic HO-1 levels were significantly higher in the lithium group than in the SCI group (P < 0.05). Additionally, immunofluorescence staining showed that the numbers of nuclear Nrf2- and cytoplasmic HO-1-positive cells were significantly higher in the lithium group than in the SCI group (Figure 5C), indicating that treatment with lithium may reduce oxidative stress in injured spinal cord tissue through the Nrf2/HO-1 pathway.
Figure 6｜Lithium attenuates inflammatory, oxidative, and pyroptotic injury in PC12 cells subjected to OGD.
IL-1β, IL-6, and TNF-α play an important role in inflammation (Schneider et al., 2019). IL-1β, IL-6, and TNF-α expression levels were significantly increased after the cells were subjected to OGD, and treatment with lithium significantly downregulated their expression compared with the OGD group. This effect was reversed by ATRA, an Nrf2 inhibitor, although inflammatory factor expression did not recover to the levels seen in the OGD group (P < 0.05; Figure 6A–C).
A number of indicators were evaluated to determine the level of oxidative stress in PC12 cells subjected to OGD. In the OGD group, CAT, GSH-Px, SOD, and T-AOC levels were decreased, and MDA and LDH levels were significantly increased compared with the control group. Compared with the OGD group, cells pretreated with lithium exhibited decreased levels of MDA and LDH but increased levels of GSH-Px, SOD, CAT, and T-AOC. However, there were no significant differences in T-AOC, CAT, or LDH expression levels between the ATRA and LiCl groups (P < 0.05; Figure 6D–I).
PI and Annexin V double-staining was used to label pyroptotic cells (Figure 6J–N, upper right quadrant) (Miao et al., 2010). The pyroptosis rate in the OGD group was markedly higher than that in the control group. In addition, the lithium and ATRA groups exhibited lower pyroptosis rates than the OGD group. Next, flow cytometric analysis of Annexin V–positive and PI-negative staining was used to identify apoptotic cells (Chi et al., 2020). There was no significant difference in pyroptosis rates among the OGD, LiCl, and ATRA groups (Figure 6J–N, upper left quadrant; P < 0.05). Taken together, these findings indicate that lithium treatment has a vital neuroprotective effect in protecting cells against OGD-induced pyroptosis.
Figure 7｜Lithium attenuates pyroptotic injury by activating the Nrf2/HO-1 signaling pathway in PC12 cells subjected to OGD.
Western blotting was used to assess the expression levels of some key proteins involved in pyroptosis. NLRP3, ASC, pro-Caspase-1, Caspase-1, GSDMD, and IL-18 expression levels were markedly increased in the OGD group compared with the control group. This effect was largely reversed by lithium. The expression level of all pyroptosis-related proteins, with the exception of pro-Caspase-1, was significantly increased in the ATRA group compared with the lithium group (P < 0.05; Figure 7A and B). Therefore, we concluded that lithium inhibits pyroptosis by downregulating NLRP3, ASC, pro-Caspase-1, Caspase-1, GSDMD, and IL-18 expression.
To investigate the possible mechanism by which lithium protects PC12 cells from pyroptosis, we assessed changes in Nrf2 and HO-1 expression levels induced by OGD. Western blotting showed that treatment with lithium greatly increased the levels of Nrf2 and HO-1 compared with the OGD group, and this effect was significantly reversed by treatment with ATRA (P < 0.05; Figure 7C and D). These findings were confirmed by immunofluorescence (Figure 7E and F).