脑损伤
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Figure 1｜LZ-3 reduces infarct volume and neuronal damage in rats post-stroke.
For the in vivo experiments, LZ-3 (Top-peptide, Shanghai, China; Figure 1A) was dissolved in 0.9% normal saline to concentrations of 1, 2, and 4 mg/ml before injection. For the in vitro experiments, LZ-3 and upadacitinib (a JAK1 inhibitor; Ark Pharm, Chicago, IL, USA) were dissolved in dimethyl sulfoxide to a concentration of 100 mM and stored at 4°C, and the experiments were performed by 1000-fold dilution of the drug to 100 μM in the cell growth medium. The final dimethyl sulfoxide concentration added to the culture medium was 0.1% (v/v), which had no significant effect on cell proliferation.
Neurological scores were determined 24 hours after reperfusion. Rats with a neurological score of 0 (no neurological deficits or abnormal activity) were excluded. Rats with neurological scores between 1 and 4 were randomly assigned to the different treatment groups (n = 30 rats per group); the mean neurological scores of each group were similar. The sham group was injected with normal saline (3 mL/kg), the edaravone group was injected with edaravone (a brain protectant (free radical scavenger); Sinopharm, Beijing, China) at a dose of 6 mg/kg, and the LZ-3 groups were injected with LZ-3 (1, 2, or 4 mg/kg). The treatments were administered by tail vein injection every 24 hours from day 1 to day 7. The in vivo experimental design is shown in Figure 1B.
The 2,3,5-triphenyte-trazoliumchloride staining results showed edaravone and LZ-3 (2 and 4 mg/kg) decreased infarct volume compared with the model group on day 3 (Figure 1C and D). To confirm the neuroprotective effect of LZ-3, we preformed immunofluorescence staining of neurons in the rat cortex in the early phase after stroke (Figure 1E) and found that LZ-3 (2 and 4 mg/kg) and edaravone (6 mg/kg) significantly increased the number of NeuN+ neurons on day 3 after stroke compared with no treatment (Figure 1F). Collectively, these results indicate that LZ-3 can reduce the damage to the brain cortex caused by stroke.
Figure 3｜Effect of LZ-3 on brain histopathology in rats on day 26 after cerebral ischemia.
After the final Morris water maze test was completed, the rats were sacrificed, and the brain tissue was evaluated by hematoxylin-eosin staining. As shown in Figure 3A, severe liquefaction necrosis was observed in the cortex in the model group, and the structure of nerve cells in the cortical area was destroyed, while LZ-3 and edaravone reduced cortical voids and improved the structural integrity of cortical nerve cells. Moreover, to evaluate the effect of LZ-3 on cortical nerve cell apoptosis after cerebral ischemia, apoptotic bodies were observed by TUNEL staining (Figure 3B). We did not observe any apoptotic bodies in the cortex of sham-operated rats, and LZ-3 and edaravone significantly reduced the number of TUNEL+ cells in the cortex post-stroke compared to the model group (Figure 3C). Then, we evaluated the effect of LZ-3 on the structural integrity of the hippocampal CA1 region of rats post-stroke by Nissl staining (Figure 3D). In comparison to the model group, LZ-3 increased the number of hippocampal neurons and hippocampal structure integrity post-stroke (Figure 3E). These results indicate that LZ-3 treatment promotes cortical and hippocampal functional recovery in rats with ischemic brain injury.
Figure 6｜Effect of LZ-3 on microglia polarization in vivo and in vitro.
Next, immunofluorescence staining was performed to observe microglia/macrophage polarization in the rat cortex. The results showed that, compared with model group, treatment with LZ-3 or upadacitinib reduced the number of ischemia-activated iNOS+ and Iba-1+ microglia/macrophage cells (Figure 6A and B) and increased the number of CD206+ and Iba-1+ microglia/macrophage cells (Figure 6C and D). To analyze whether LZ-3 regulates microglia/macrophage polarization in vitro and in vivo, we detected polarization-related levels of marker mRNAs in BV2 cells by qPCR. As shown in Figure 6E and F, LZ-3 and upadacitinib reduced the mRNA levels of M1 markers (CD11b, CD32, and iNOS) compared with the model group. In addition, LZ-3 promoted microglia polarization toward the M2 phenotype, as indicated by increased mRNA levels of M2 markers (Arg-1, IL-10, and CD206). These findings show that LZ-3 affects M1- and M2-type polarization, and that the mechanism of this effect may be related to inhibition of JAK1.
Figure 7｜ LZ-3 decreases microglia phagocytosis and migration after ischemia or hypoxia.
To determine whether LZ-3 regulates microglial functions such as phagocytosis and migration after ischemia, the ability of microglia to phagocytose neurons in the cortex was observed by immunofluorescence staining (Figure 7A). We found that LZ-3 treatment reduced the microglial phagocytosis induced by ischemia, as well as the number of neurons (NeuN-positive cells) phagocytosed by microglia, compared with no treatment (Figure 7B). In addition, microglial migration was assessed by cell migration assay, which showed that treatment with LZ-3 or upadacitinib reduced the BV2 cell migration rate compared with the OGD group (Figure 7C and D). These findings demonstrated that LZ-3 inhibits ischemia-induced phagocytosis and microglia migration.