The current study demonstrated that DM enhanced the impairment of neurobehavioral performance compared to NG after I/R as reflected by the higher deficit score in DM than that of the NG animals. The worsened neurological deficit in DM animals was associated with enlarged infarct volume and increased number of neural damage in the hippocampal CA1 area. Treatment by rapamycin to DM ischemic animals improved neurological functional performance and reduced infarct volume and neuronal death. Therefore, rapamycin could be considered as therapeutic drug for ischemic patients accompanied with diabetes mellitus.
Rapamycin and its derivatives (such as everolimus and tacrolimus) are specific inhibitors of m-TOR (Ruiz-Torres et al. 2018). Rapamycin binds to intracellular receptor FKBP12 and directly acts on FRB (fkbp-12-rapamycin binding) domain in mTOR, thereby inhibiting the activity of mTORC1 kinase (Sun et al. 2018). Whether activation of mTOR pathway is detrimental or beneficial to ischemic injury remains controversial. For example, activating PI3K/Akt/m-TOR by MiR-27 activator or Hsp90 silencing has been shown to protect rat brain from ischemia and reperfusion damage, and to suppress oxidative stress and inflammatory response. In contrary, other studies have also demonstrated that inhibition of mTOR reduced nerve injury and promoted neuronal survival (Zhang et al. 2020). These contradictory results suggest the importance of delicate balance of mTOR signaling pathway in cerebral ischemia. Although whether mTOR is beneficial to ischemic injury remains a debate, it is undeniable that rapamycin, its inhibitor, has dominantly shown a protective effect in many nervous system diseases (Brandt et al. 2018;Hwang et al. 2017;Liu et al. 2016;Yang et al. 2015;Yang et al. 2019). In this study, we have observed that DM significantly increased the levels of p-mTOR and p-S6 levels after I/R and the neuroprotective effect of rapamycin in DM animals was linked with suppression of p-mTOR and p-S6, supporting the concept that inhibition of m-TOR signaling protects the brain from ischemic damage. This is consistent with the results we have previously reported in rats subjected to global ischemia under hyperglycemic condition and normoglycemic conditions (Liu et al. 2016;Yang et al. 2015). The mechanisms mediating the protective effect of rapamycin may include modulation of PI3K/Akt, MAPK/ERK1/2, and autophagy signaling (Yang et al. 2019) .
Mitochondria play an important role in promoting neural survival and improving neural functional recovery after ischemic stroke. As highly dynamic organelles, mitochondria undergo continuous fission and fusion to maintain mitochondrial function and cell activity. Mitochondrial fission contributes to removal of damaged and redundant mitochondria. Mitochondrial fission is considered as a prerequisite for mitochondrial phagocytosis. Mitochondrial fusion facilitates exchange of content between mitochondria and regulates mitochondrial metabolism (Huang et al. 2020). The balance between mitochondrial fission and fusion is regulated by a group of dynamic related large GTPases located in the outer membrane and inner membrane of the mitochondria. Fission is driven by DRP1 and Fis1; while fusion is driven by OPA1, and Mfn1 and Mfn2 (Singh et al. 2017). It has been reported that cerebral ischemia suppresses the levels of fusion regulators OPA1 and Mfn2, and increases the levels of division regulators DRP1 and Fis1. Excessive fission results in mitochondrial rupture and fragmentation (Yoo and Jung 2018). Our results showed that DM further increased the levels of p-DRP1, suggesting increased probability of excessive mitochondrial fission by diabetic hyperglycemia. The small scale increase of OPA1 in DM rats after I/R may represent a nature response by which mitochondria try to combat the excessive fission. Rapamycin profoundly inhibited the levels of p-DRP1 and increased the level of OPA1, suggesting the imbalance of mitochondrial fission and fusion could be prevented by rapamycin. It is likely that hyperglycemia further tilts mitochondrial dynamic balance towards fission and rapamycin is capable to prevent or reverse this dynamic imbalance.
SIRT3 is a deacetylate enzyme containing a variety of amino acids. It is a member of sirtuins family and a redox enzyme. SIRT3 has been shown to decrease cell death and tissue damage through blockade of mitochondrial permeability transition pore formation through deacetylation of cyclophilin D in the mitochondrial matrix, to activate mitochondrial biogenesis through deacetylation of PGC-1α, to regulate cell metabolism by modulating AMPK (Yu et al. 2019), and to inhibit the activity of m-TOR (Wang et al. 2021). There has been very few study reports the expression of SIRT3 in diabetic ischemic models. In current study, SIRT3 increased after ischemia and reperfusion in NG animals. DM inhibited this ischemia-caused increase; while rapamycin abolished the inhibitory effect of DM on SIRT3, suggesting that SIRT3 plays a protective role after ischemic brain damage.
Nix/BNIP3L locates in the outer membrane of mitochondria. Studies have shown that BNIP3L deficiency significantly aggravates ischemic brain injury (Wu et al. 2021), suggesting that BNIP3L can protect nerve cells. In current study, compared with NG group, DM significantly reduced the protein level of Nix/BNIP3L, and rapamycin treatment partially reversed its decline, suggesting that Nix/BNIP3L may have a protective effect against ischemic injury.
In conclusion, diabetic hyperglycemia aggravated brain damage caused by MCAO. This enhanced damage was associated with activations of p-mTOR, autophagy, mitochondrial fission and suppressions of SIRT3 and Nix/BNIP3L. Rapamycin prevented the DM enhanced ischemic brain damage, suppressed p-mTOR, rebalanced mitochondrial dynamics, and increased the levels of neural survival factors SIRT3 and Nix/BNIP3L.