IF attenuates hemorrhagic brain damage in mice
To determine the effects of IF on ICH-induced brain damage, mice were assigned randomly to ICH and ICH+IF groups and sacrificed at 1, 3, 7 and 28 days after ICH onset. Compared with ICH groups, the motor function of mice was improved significantly on an IF diet except for the day 1 (Tab S1, S2, Fig 1A, B). Mice treated with IF had smaller hematoma than ICH groups at day 3 and 7 after ICH induction (Fig 1C, D). Consistently, IF reduced brain edema significantly as indicated by hemisphere volumes at day 3 and 7 except for day 1 (Tab S3, Fig 1C, E). At day 28, IF-treated mice showed reduced striatum atrophy as indicated by DARPP-32 positive area shrinkage as compared with ICH groups (24.76%±5.36% vs 18.34%±3.59%, n=6, p<0.05, Fig 1F, G). TUNEL staining showed that the number of apoptotic cells was reduced at day 1, 3 and 7 (Tab S4, Fig 1H, I). On the other side, IF preserved the number of Nissl-positive neurons in the ipsilateral hemisphere significantly from day 1 to 28 after ICH (Tab S5, Fig 1J, K). These results indicated that IF had protective effects against brain damage and neurological deficits in both the acute and chronic stages of ICH in mice.
IF regulates microglia activation and inflammatory cytokines release after ICH
We next investigated the effects of IF on microglia activation after ICH. Iba-1 positive microglia accumulated at day 1 in the ipsilateral hemisphere, increased from day 3 to 7, but decreased at day 28 (Fig 2A, B). Interestingly, IF significantly reduced the number of Iba-1+ microglia at day 3 and 28 but increased it at day 7 (Tab S6, Fig 2A, B). However, microglia-driven inflammatory response measured with ELISA indicated that proinflammatory factors IL-1β and TNF-α in the blood were increased in response to ICH, which were attenuated by IF-treatment from day 3 to day 28 (Tab S7, Fig 2C, D). In addition, the mRNA levels of IL-1β and TNF-α in brain tissue around hematoma also increased after ICH, which was attenuated by IF (Tab S8, Fig 2E, F). Considering the plasticity of microglia in response to different microenvironment, we speculated that the inconsistency in changes of microglia activation and proinflammatory factors secretory was possibly due to different functional types of microglia. As shown in Fig 2G-J, the proinflammatory marker CD16 and anti-inflammatory marker Arg1 were double stained with Iba-1 at day 3 and 7 respectively. IF significantly reduced the CD16+Iba-1+ cells at day 3 (202.00±48.00 vs 85.00±32, n=12, p<0.01, Fig 2G, I) and increased Arg1+Iba-1+ at day 7 (106.00±35.00 vs 254.00±48.00, n=12, p<0.01, Fig 1H, J) after ICH onset. These results indicated that IF might suppress microglia-induced inflammation in acute stage and drive microglia polarization towards anti-inflammation phenotype in subacute stage of ICH.
Microglial Sirt3 is upregulated by IF after ICH
We further investigated whether Sirt3 is involved in the effects of IF on microglia activation after ICH. Immunofluorescence staining was used to offer direct evidence of Sirt3 expression in microglia. As shown in Fig 3A and B, Sirt3 expression was increased in microglia at 3 days after ICH as compared to Sham groups, which further increased in ICH+IF group (sham 8.35±2.24 vs ICH 12.62±4.26 vs ICH+IF 18.81±3.17, n=12, p<0.01). Similar results were also obtained for day 7 (ICH 12.45±8.76 vs ICH+IF 21.23±6.33, n=12, p<0.05, Fig S1). In addition, western blot also confirmed the upregulated Sirt3 in ICH and ICH+IF groups (Sham 1.00±0.08 vs ICH 1.13±0.22 vs ICH+IF 1.52±0.12, n=5, p<0.01, Fig 3C, D). These data indicated that Sirt3 in microglia was upregulated by IF, and might play a role in IF-induced neuroprotection after ICH.
Microglial Sirt3 deletion attenuates the neuroprotective effects of IF following ICH
Sirt3f/f;Cx3cr1-Cre (Sirt3 cKO) mice in which Sirt3 was deleted specifically in microglia were used to determine the role of microglial Sirt3 in IF-induced neuroprotective response. As shown in Fig 4A and B, Sirt3 cKO mice exhibited worse motor functions as indicated by the higher corner turn and forelimb use asymmetry scores as compared to control Sirt3f/f mice in both the acute and chronic stages after ICH+IF (Tab S1, S2). In addition, Sirt3 cKO mice had larger hematoma volume and edema content (Tab S3, Fig 4C-E) at day 3 and 7 and more severe striatum atrophy at day 28 as compared to control Sirt3f/f mice after ICH+IF (Sirt3f/f 17.45%±4.26% vs Sirt3 cKO 25.32%±5.18%, n=6, p<0.05, Fig 4F, G). TUNEL staining showed that at day 1 after ICH, the number of TUNEL-positive neurons was higher in Sirt3 cKO group (Tab S4, Fig 4H, I). These data suggested that microglial Sirt3 is required for IF-induced neuroprotective effects after ICH.
Sirt3 contributes to anti-neuroinflammatory effects of IF in the acute stage of ICH
The role of Sirt3 in regulating microglia-driven inflammatory response in ICH+IF in the acute stage of ICH (day 3) was further investigated. At day 3 after ICH+IF, the number of Iba-1+ microglia in the perihematomal area was greater in Sirt3 cKO than in Sirt3f/f mice ( Sirt3f/f 226.00±36.00 vs Sirt3 cKO 327.00±43.00, n=6, p<0.01, Fig 5A, B). Accordingly, the mRNA levels of IL-1β (Sirt3f/f 1.00±0.23 vs Sirt3 cKO 2.13±0.47, n=3, p<0.05, Fig 5C) and TNF-α (Sirt3f/f 1.00±0.18 vs Sirt3 cKO 2.87±0.75, n=3, p<0.05, Fig 5D) in perihematomal areas were higher in Sirt3 cKO mice than in Sirt3f/f counterparts. ELISA analysis of the serum also showed higher levels of IL-1β (Sirt3f/f 109.52±16.28 vs Sirt3 cKO 164.33±23.32 pg/ml, n=6, p<0.01, Fig 5E) and TNF-α (Sirt3f/f 6.06±3.12 vs Sirt3 cKO 9.67±2.23 pg/ml, n=6, p<0.05, Fig 5F) in Sirt3 cKO mice. Western blot also showed that the proinflammatory markers CD16 (Sirt3f/f 1.00±0.09 vs Sirt3 cKO 1.43±0.12, n=3, p<0.01, Fig 5G, H) and CCL3 (Sirt3f/f 1.00±0.09 vs Sirt3 cKO 2.13±0.21, n=3, p<0.01, Fig 5G, I) were significantly increased in microglia isolated from perihematomal regions in Sirt3 cKO mice as compared to Sirt3f/f mice.
Microglial Sirt3 promotes microglia polarization to anti-neuroinflammatory phenotype in the subacute stage of ICH
Because IF increased Arg+Iba-1+ cells while reduced proinflammatory factor release in the subacute phase (day 7) after ICH, there parameters were further measured in Sirt3 cKO mice. The number of Iba-1+ microglia had limited change in the perihematomal area was not different between Sirt3f/f mice and Sirt3f/f cKO mice at day 7 treated with IF after ICH (Sirt3f/f 532.00±83.00 vs Sirt3 cKO 473.00±73.00, n=6, p=0.22, Fig 6A, B). However, the mRNA levels of IL-10 (Sirt3f/f 1.00±0.15 vs Sirt3 cKO 0.41±0.27, n=3, p<0.05, Fig 6C) and TGF-β1 (Sirt3f/f 1.00±0.15 vs Sirt3 cKO 0.62±0.18, n=3, p<0.05, Fig 6D) in perihematomal areas were lower in Sirt3 cKO mice than in Sirt3f/f mice. Consistently, levels of IL-10 (Sirt3f/f 15.32±3.16 vs Sirt3 cKO 1.73±0.86, n=6, p<0.01, Fig 6E) and TGF-β1 (Sirt3f/f 168.44±31.70 vs Sirt3 cKO 107.23±14.52, n=6, p<0.01, Fig 6F) in serum also decreased in Sirt3 cKO mice. In addition, the anti-inflammatory markers CD163 (Sirt3f/f 1.00±0.14 vs Sirt3 cKO 0.54±0.25, n=3, p<0.05, Fig 6G, H) and CCL22 (Sirt3f/f 1.00±0.17 vs Sirt3 cKO 0.61±0.16, n=3, p<0.05, Fig 6G, I) were significantly downregulated in microglia in the perihematomal regions in Sirt3 cKO mice as compared to Sirt3f/f mice. These observations suggest that microglial Sirt3 is involved in the regulation of microglia polarization in ICH+IF.
Sirt3 regulates microglial activation through Nrf2/HO-1 pathway in acute stage of ICH
To investigate whether the Nrf2/HO-1 signalling pathway was regulated by Sirt3 in ICH+IF, the protein levels of Nrf2 and HO-1 were first measured at multiple timepoints in the acute and subacute stages of ICH in WT mice. As shown in Fig S2, both Nrf2 and HO-1 proteins increased significantly at day 1 and 3 but decreased almost to the baseline levels at day 7. Therefore, we chose day 3 as the timepoint for further study. At day 3 after ICH, IF upregulated the expressions of Nrf2 and HO-1 in microglia which were attenuated by Sirt3 deletion (Fig 7A-C). Consistently, IF-induced increases in number of HO-1+ cells were attenuated by Sirt3 deletion (Fig 7D, E). Next, Sirt3f/f cKO mice were pre-treated with a clinically approved Nrf2 activator, dimethyl fumarate (DMF) or vehicle control before ICH+IF. HO-1 (1.00±0.21 vs 1.44±0.16, n=3, p<0.05, Fig 7F, G) and Nrf2 (1.00±0.16 vs 1.87±0.20, n=3, p<0.05, Fig 6F, H) levels were increased markedly in the ICH+IF+DMF groups as compared to ICH+IF+Vehicle groups. Moreover, the secretion of IL-1β (164.33±23.32 vs 138.25±15.33, n=6, p<0.05, Fig 7I) and TNF-α (9.67±2.23 vs 7.04±1.76, n=6, p<0.05, Fig 7J) in serum were partly reduced by DMF. Altogether, these results indicated that the Nrf2/HO-1 signalling pathway may be downstream of Sirt3 in microglial activation at day 3 after ICH.