SPC Promoted Mouse Primary Microglia Polarization into the Anti-Inflammatory M2 Phenotype and Elicited A Protective Effect against OGD
As illustrated in Fig. 1A left panel, OGD increased the lactate dehydrogenase (LDH) release to 496.4 (14.91%) compared to that of the control group (P < 0.0001), and SPC showed a significant effect on the prevention of increased LDH levels induced by OGD [357.8 (39.68%) in the Sevo + OGD group, P = 0.0046]. Compared with that in the control group, the cell viability in the OGD group was significantly reduced. SPC restored the cell viability [figure 1A, right panel, 36.42 (1.66%) in the OGD group vs. 74.37 (3.33%) in the Sevo + OGD group, P = 0.0037].
As shown in Fig. 1B, the proinflammatory factor (TNF-α, IL-1β, iNOS) mRNA levels in the OGD group were higher than those in the control group. When the mouse primary microglia cells were pre-treated with sevoflurane, the mRNA expression was decreased compared with that of the OGD group (P = 0.0023, < 0.001, 0.0073). The mRNA changes of anti-inflammatory mediators are presented in Fig. 1C. Treatment with OGD elevated the CD206, YM1/2 and arginase-1 mRNA levels compared with those in the control group (P < 0.001), while SPC prevented this increase in mRNA expression (P = 0.0019, < 0.001, and 0.007, respectively).
SPC Promoted Mouse Primary Microglial Polarization into the Anti-Inflammatory M2 Phenotype and Protected Cells against LPS-Induced Injury
LPS stimulation was also performed in this study to further determine the effect of SPC on microglia polarized to the M2 phenotype (Fig. 1D-F). As shown in Fig. 1D, the microglial culture was severely damaged by LPS stimulation, which was demonstrated by the increased LDH release and decreased MTT levels in the LPS group (P < 0.001). This toxicity was reduced when the microglial cells were pre-treated with sevoflurane (P = 0.0023 for LDH, P = 0.022 for MTT).
The LPS treatment significantly up-regulated the mRNA levels of M1 marker genes (TNF-α, IL-1β, and iNOS), while SPC exerted a significant effect on the inhibition of LPS-induced elevation of these proinflammatory genes (P = 0.0033, < 0.001, and 0.0015, respectively). In addition, SPC treatment improved the mRNA levels of M2 marker genes (CD206: P = 0.0057, YM1/2: P = 0.0056, arginase-1: P = 0.0037).
SPC Reduced Proinflammatory Factor Expression and Enhanced M2 Microglia/Macrophage Polarization in The Ischemic Hemisphere of The Mice at 7 and 14 Days After Reperfusion.
To demonstrate the correlation of SPC and M2 polarization in vivo, we measured the mRNA levels of proinflammatory and anti-inflammatory factors at 7 (Fig. 2A-B) and 14 (Fig. 2C-D) days after reperfusion in a transient MCAO model.
Changes in physiologic parameters at the end of the preconditioning operation and various intervals of I/R are summarized in Supplementary Tab. S1. No significant differences in the pH value, temporal temperature, or partial pressure of carbon dioxide (PCO2) were detected among the groups. As shown in in Supplementary Fig. S1, SPC did not alter the regional cerebral blood flow.
As presented in Fig. 2A, the TNF-α, IL-1β and iNOS mRNA levels were increased at 7 days after I/R, while this elevation was reversed by pretreatment with 5 days of sevoflurane (P = 0.0202, 0.006, and < 0.001, respectively). As shown in Fig. 2B, I/R did not alter the mRNA expression of anti-inflammatory mediators (CD206: P = 0.6507, YM1/2: P > 0.9999, arginase-1: P = 0.7854). However, the mRNA levels of the anti-inflammatory mediators in the SPC-treated group were significantly increased compared with those of the I/R group (P = 0.0006, P = 0.0013, P = 0.0012).
Consistent with the results at 7 days after reperfusion, the mRNA expression of these proinflammatory factors was increased in the infarct penumbra at 14 days after reperfusion (Fig. 2C). However, the increased mRNA levels of TNF-α, IL-1β and iNOS were also prevented by SPC at this time-point (P = 0.0006, 0.0013, and 0.0012, respectively). As shown in Fig. 2D, no significant change in the mRNA levels of M2 marker genes was detected between the control and I/R groups (control vs. I/R: P = 0.6081, 0.2765, 0.1144), while SPC significantly increased the mRNA expression of these anti-inflammatory factors (I/R vs. Sevo + I/R: P < 0.001, = 0.002, < 0.001).
Sevoflurane Preconditioning Increased the Phosphorylation of GSK-3β, and Supplementation With A GSK-3β Inhibitor Reduced Cerebral I/R Injury
As shown in Fig. 3A, the GSK-3β phosphorylation was determined by Western blot analysis and immunofluorescence staining. The abundance of phosphorylated GSK-3β in the I/R groups was reduced compared with that of the control group (P = 0.0218), but this reduction was ameliorated in the SPC group (Sevo + I/R vs. I/R, P = 0.0206). Neither I/R nor Sevo + I/R treatment affected the total GSK-3β protein expression. As expressed in the right panel of Fig. 3A, the results of double immunofluorescence staining demonstrated that increased GSK-3 phosphorylation produced by SPC was mainly colocalized with neurons.
To further verify the role of GSK-3β in I/R tolerance induced by SPC, we used a GSK-3β inhibitor, TDZD, in this study. As shown in Fig. 3B, SPC significantly improved the neurobehavioral outcome, as judged by the neurological score, at 1 day [10.75, (9.00, 12.00)], 2 days [10.00 (7.00, 11.00)] and 3 days [9.50, (7.00, 11.00)] in the Sevo + I/R group compared with the I/R group at parallel time-points (P = 0.0031, 0.0121, and 0.0008, respectively, for each comparison). Consistent with the improvement in neurological outcome, the infarct volume in the Sevo + I/R group was smaller than that in the I/R group [figure 3C, 30.5 (2.9%) vs. 43.1 (3.1), P = 0.0142). Following the administration of TDZD, mice that underwent the MCAO surgery showed better neurobehavioral performance than I/R mice (P = 0.0171, 0.0241, 0.0219 in 1, 2 and 3 days after reperfusion, respectively). Additionally, supplementation with TDZD reduced the brain infarct volume compared with that of the I/R mice [28.9 (1.9%) vs. 43.1 (3.1%), P = 0.0053]. No statistically significant difference was detected between the I/R and I/R + vehicle groups.
Neuronal cell apoptosis in the ischemic penumbra is shown in Fig. 3D. At 72 h after reperfusion, the number of TUNEL-positive cells in the SPC group was significantly decreased compared with that of the I/R group (P = 0.0186). Moreover, the number of TUNEL-positive cells in the TDZD-treated group was reduced compared to that in the I/R + vehicle group (P = 0.0314).
GSK-3β Inhibition Promoted M2 Microglia/Macrophage Polarization in the Ischemic Hemisphere at 7 Days After Reperfusion
As shown in Supplementary Fig. S2, the GSK-3β phosphorylation was measured by Western blots. The phosphorylation of GSK-3β at Ser9 was decreased in the vehicle-treated I/R groups compared with the control group (P = 0.0210), but this reduction was ameliorated by TDZD supplementation (TDZD + I/R vs. I/R + vehicle, P = 0.0045).
As illustrated in Fig. 4, the protein (Fig. 4A) and mRNA (Fig. 4B) levels of TNF-α, IL-1β and iNOS were examined by ELISA kits. The protein levels of these proinflammatory factors were exaggerated in the I/R group compared with the control group but was reduced in the SPC group (P = 0.0279, 0.0054, 0.0170, respectively). The administration of TDZD also reduced the expression of these M1 markers compared with those of the I/R group (P = 0.0078, 0.0030, 0.0049, respectively). No significant difference was detected between the TDZD + I/R and Sevo + vehicle groups. As shown in Fig. 4B, the mRNA expression of the M1 proinflammatory factors was significantly elevated in the ischemic penumbra but was reduced when mice received SPC. The TDZD treatment also reduced the elevated mRNA expression of these proinflammatory factors (TDZD + I/R vs. I/R: P = 0.0206, < 0.001, 0.0157, respectively). As shown in Fig. 4C, the mRNA levels of the M2 gene markers in the Sevo + I/R group were higher than those in the I/R group. TDZD also increased the level of these anti-inflammatory factors (CD206, YM1/2, arginase-1: P < 0.001).
SPC and TDZD Treatment Increased Nuclear Translocation of Nrf2, And Nrf2 Deficiency Reversed the Neuroprotective Effect Produced by TDZD Supplementation
As shown in Fig. 5A, the Nrf2 protein content in the nucleus was increased in the SPC and TDZD-treated groups compared to the I/R group (P = 0.0222 and 0.0323, respectively). As shown in Fig. 5A (right panel), double immunofluorescence staining showed that the Nrf2 protein was increased by SPC and TDZD treatment, and Nrf2-positive cells were mainly neurons.
To further determine the relationship between GSK-3β and Nrf2 under SPC, we applied an Nrf2-shRNA (AAV-Nrf2 and its control AAV-GFP) in this experiment to silence the expression of Nrf2. As shown in Fig. 5B, TDZD significantly ameliorated the neurobehavioral outcome, while Nrf2 knockdown reversed this improvement, as judged by the neurological scores at 1 day, 3 days and 7 days in the TDZD + AAV-Nrf2 group compared with the TDZD group at parallel timepoints (P = 0.0193, 0.0219, and 0.0132, respectively, for each comparison). Consistent with the change in neurological outcome, the infarct size in the TDZD + AAV-Nrf2 group was larger than that in the TDZD group at 7 days after reperfusion [figure 5C, 32.51 (1.51%) vs. 24.34 (1.2%), P = 0.0049]. However, no significant difference was detected between the I/R and I/R + vehicle groups. At 72 h after reperfusion, the reduced number of TUNEL-positive cells in the TDZD group was significantly reversed by the introduction of the Nrf2 mutation (Fig. 5D, P = 0.0265).
Knockdown of Nrf2 Abolished the Promoted M2 Microglia/Macrophage Polarization Produced by GSK-3β Inhibition
AAV-Nrf2 microinjection led to a significant reduction of Nrf2 positive cells (see Supplementary Fig. S3). The protein levels of these proinflammatory factors were reduced in the TDZD group compared with the I/R group, while Nrf2 knockdown prevented this reduction (Fig. 6A; P = 0.0241, 0.0183, 0.0081). As shown in Fig. 6B, the mRNA expression of the M1 proinflammatory factors was significantly reduced in the ischemic penumbra treated with TDZD; however, this attenuated expression was abolished by the administration of AAV-Nrf2 (P = 0.0073, 0.0407, 0.0205, respectively). As shown in Fig. 6C, the mRNA levels of anti-inflammatory factors were higher in the TDZD group than in the I/R group, while Nrf2 deficiency prevented this improvement (CD206, YM1/2, and arginase-1: P < 0.001, = 0.0150, and 0.0127, respectively).
Knockdown of Nrf2 Reversed M2 Microglia/Macrophage Polarization and Abolished The Inhibition of Reactive Oxygen Species Generation Produced by TDZD Treatment
Representative images of flow cytometry that were used to examine the ratio of M1- and M2-positive microglia are shown in Fig. 7A. As shown in Fig. 7B, activated M1 microglia (CD86+) intensely accumulated in the ischemic penumbra compared with the ipsilateral hemisphere of the control group (P < 0.001), while SPC and TDZD treatment significantly reduced this accumulation (P < 0.001 and P = 0.0003, respectively). However, this reduction was reversed by mutation of Nrf2 (P = 0.003). As shown in Fig. 7C, the percentage of M2-positive microglia (CD206+) was not affected by I/R surgery, but both SPC and TDZD treatment increased this ratio compared with that of the I/R group (Sevo + I/R: P < 0.001, TDZD + I/R: P = 0.0013). As expected, supplementation with AAV-Nrf2 reversed the improved accumulation of M2 microglia (P = 0.016). No significant difference was detected between the TDZD and TDZD + AAV-GFP groups in the percentage of M1 or M2 microglia.
As indicated in Fig. 7D, the generation of reactive oxygen species (ROS) was measured by DHE staining. Increased levels of DHE-positive cells were detected in the I/R group, while both SPC and TDZD treatment reduced this increase (Fig. 7E, Sevo + I/R: P = 0.0257, TDZD + I/R: P = 0.0323). Moreover, mutation of Nrf2 reversed the reduced DHE-positive cells induced by TDZD (P = 0.0131).