Background:At present, ischemic stroke has become one of the most serious diseases affecting human health, bringing serious burden to families and society. However, reperfusion of blood flow can cause secondary injury, cerebral ischemia/reperfusion injury (CI/RI).
Objective: To investigate whether the nanoparticles composed of EXOsomes coated with the diosmetin have effects on cerebral ischemia-reperfusion injury C57BL/6J mice and oxygen-glucose deprivation/re-oxygenation. The protective effect of OGD/R induced SH-SY5Y cells and the related mechanism were discussed, which provided experimental basis and theoretical basis for the clinical application of geranyl in the prevention and treatment of ischemic stroke and other ischemic.
1.Characterization of EXO and EXO+Dios
Methods: (1) Serum EXOsomes of SD rats were extracted by kit. (2) The morphology of EXO and EXO+Dios was observed by TEM, the Zeta potential of EXO and EXO+Dios was detected by particle size analyzer, the particle size of EXO and EXO+Dios was detected by NTA, and CD9, ALIX, TSG101 and Calnexin were detected by WB Four proteins were identified by EXO and EXO+Dios. Geranylin in EXO was determined by high performance liquid chromatography (HPLC).
Results: The EXOsomes observed under TEM are spherical structures with a diameter of about 100 nm, which is consistent with the diameter distribution range of EXOsomes in the guidelines. The morphology of EXOsomes loaded with geranophyllin did not change significantly under TEM, but was still round and spherical. The EXO+Dios observed under TEM after drug loading was about 150nm in straight size, with an increase in diameter and no significant change in morphology. The particle size distribution of EXOsomes detected by NTA was 129nm on average, and the particle size of EXOsomes increased slightly after drug loading to about 135nm. Both EXOsomes and baicalein EXOsomes expressed CD9, ALIX, and TSG101, indicating that the protein content of EXOsomes had no significant change before and after drug loading. The encapsulation rate EE and loading rate LR of EXO+Dios were 44.21% and 8.12% respectively.
2.Protective effect and mechanism of Diosmetin and EXO+Dios on cerebral ischemia-reperfusion mice
Methods: (1) 60 male SD mice were randomly divided into 5 groups: Sham group, model group (MCAO/R group), EXOsome group (EXO group), geraniol treatment group (Dios group), geraniol + EXOsome group (EXO+Dios group); Three drug groups were given corresponding drug treatment, sham operation group and model group were given the same amount of normal saline. One hour after the last dose, the mice underwent right middle cerebral artery occlusion (MCAO) modeling, 1.5h ischemia, and 24h reperfusion. (2) All mice were evaluated for neurological impairment; (3) TTC dye solution was used to stain the brain sections, and Image J software was used to analyze and calculate the cerebral infarction volume of mice; (4) HE and Nissl staining were used to observe the cell morphology and NissL body injury in the ischemic cortex. (5) Endoplasmic reticulum stress related indexes (ATF4, ATF6 antibody, eIF2α, GRP78, XBP1S content, Ca2+ content detected by kit, ROS content detected by frozen section) were detected by biochemical method. (6) The expressions of GRP78, p-PERK, PERK, CHOP, ATF4, eIF2α and p-eIF2α in the ischemic cortex of mice were detected by Western blot. (7) Using graphpad prism software, statistical analysis was performed on the data and statistical charts were made.
Results: Diosmetin pretreatment can improve the neurological dysfunction, reduce the cerebellar infarct volume, and reduce the functional damage of cortical and nerve cells in cerebral ischemia-reperfusion mice. Decreased ATF4, ATF6, eIF2α, GRP78, XBP1S, Ca2+, ROS, GRP78, p-PERK, PERK, CHOP, ATF4, eIF2α and p-eIF2α; at the same time, the effect of Diosmetin on improving these indexes was more significant after EXOsome loading.
3.Protective effects and mechanisms of Diosmetin and EXO+Dios on OGD/ R-induced SH-SY5Y cells
Methods： (1) The cultured SH-SY5Y cells were randomly divided into 5 groups: the SH-SY5Y cells were divided into Control group, OGD/R group, EXOsome group (EXO), geranyllin group (Dios) and geranyllin + EXOsome group (EXO+Dios). The blank control group and OGD/R group were cultured with SH-SY5Y cell special medium after OGD for 24 h. In the EXO group, SH-SY5Y cell medium containing 36.96μg/mL EXO was added after OGD and cultured for 24 h. Diosmetin group was cultured with SH-SY5Y cell medium containing 10μM geranyl after OGD for 24 h. The EXO+Dios group was cultured with SH-SY5Y cell special medium containing 36.96μg/mL EXO+Dios (i.e., 10μM Dios) after OGD for 24h. The effects of drugs and OGD/R on the survival rate of astrocytes were detected by CCK-8 method. (2) The content of Ca2+ and ROS in each group was detected by flow cytometry. (3) ATF6 and Xbp1 were detected by Elisa. (4) The expressions of GRP78, p-PERK, PERK, CHOP, ATF4, eIF2α and p-eIF2α were detected by WB. (5) The protein expression distribution of PERK and ATF4 was detected by immunofluorescence. (6) Statistical difference analysis was performed on the data using Graphpad prism software to make statistical charts.
Results: After OGD/R, the cell viability of SH-SY5Y cells decreased, and the expressions of PERK, ATF4, p-PERK, GRP78, CHOP, p-eIF2α, eIF2α protein, Ca2+, ROS, ATF6 and XBP1 increased. Dios decreased the expression of PERK, ATF4, p-PERK, GRP78, CHOP, p-eIF2α, eIF2α protein, and decreased the expression of Ca2+, ROS, ATF6, and XBP1. EXO-loaded Dios increased the effect of Dios on reducing the expression of these indicators. The results suggest that Dios can inhibit endoplasmic reticulum stress to alleviate OGD/ R-induced SH-SY5Y cell damage, the mechanism of which may be related to the activation of PERK/ATF4 signaling pathway, and its efficacy is significantly enhanced after EXO loading.
Conclusion: Diosmetin can reduce CI/RI by inhibiting endoplasmic reticulum stress, and its mechanism may be related to the activation of PERK/ATF4 signaling pathway, and the activation of PERK/ATF4 signaling pathway is enhanced after Diosmetin is loaded by EXOsomes.
Key words: Cerebral ischemia/reperfusion injury; PERK/ATF4; Diosmetin; EXOsome; Endoplasmic reticulum stress