25-Hydroxycholesterol protecting from cerebral ischemia-reperfusion injury through the inhibition of STING activity

Abstract

Middle cerebral artery occlusion (MCAO) injury refers to impaired blood supply to the brain that is caused by a cerebrovascular disease, resulting in local brain tissue ischemia, hypoxic necrosis, and rapid neurological impairment. Nevertheless, the mechanisms involved are unclear, and pharmacological interventions are lacking. 25-Hydroxycholesterol (25-HC) was reported to be involved in cholesterol and lipid metabolism as an oxysterol molecule. This study aimed to determine whether 25-HC exerts a cerebral protective effect on MCAO injury and investigate its potential mechanism. 25-HC was administered prior to reperfusion in a mouse model of MCAO injury. 25-HC evidently decreased infarct size induced by MCAO and enhanced brain function. It reduced stimulator of interferon gene (STING) activity and regulated mTOR to inhibit autophagy and induce cerebral ischemia tolerance. Thus, 25-HC improved MCAO injury through the STING channel. As indicated in this preliminary study, 25-HC improved MCAO injury by inhibiting STING activity and autophagy as well as by reducing brain nerve cell apoptosis. Thus, it is a potential treatment drug for brain injury.

Keywords: 25-hydroxycholesterol; STING; autophagy; mTOR; middle cerebral artery occlusion.

Figure 1

25-Hydroxysterol reduces size of cerebral infarction in a mouse middle cerebral artery occlusion (MCAO) model. Mice were randomly divided into three groups. Control group, healthy mice; MCAO group, mice with MCAO; mice with MCAO treated with + 25-Hydroxymethosterol. (A) Chemical structure of 25-Hydroxysterol. (B) Neurobehavioral score and Western blot analysis of BCL2 and Bax expression to select drug concentration (**P < 0.01 versus Sham group, #P <0.05 versus MCAO group, &P<0.05 versus MCAO+25-HC 0.1mg/kg group). (C) TTC staining was used to detect cerebral infarction in mice (N = 5 per group, **P<0.001 versus sham group, #P<0.05 versus MCAO group).

Figure 2

25-Hydroxysterol reduces brain tissue apoptosis and oxidative damage and inflammation in mouse middle cerebral artery occlusion (MCAO) model. (A) Nissl staining 40× and 200× (scale bar = 50 μm/200 μm, N = 4 per group). (B) Western blots were used to detect the expressions of caspase-3, cleaved caspase-3, Bcl2, and Bax. (C–H) Serum levels of interleukin (IL-6), IL-1β, and tumor necrosis factor-α were detected by commercially available ELISA kits. Myeloperoxidase (MPO), malondialdehyde (MDA), and lactate dehydrogenase (LDH) levels in the serum were quantified by using commercial kits following instructions of the manufacturer. (N = 6 per group, **P<0.01 versus sham group, ##P<0.01, #P<0.05 versus MCAO group).

Figure 3

25-Hydroxycholesterol (25-HC) suppressed the middle cerebral artery occlusion (MCAO)-induced enhanced autophagy, and the effects of 25-HC on autophagy, oxidative injury, apoptosis, and inflammation were reversed by oxygen-10-acridine acetic acid (CMA). (A) Expression of p-mTOR, STING, P62, Beclin1, LC3 I, and LC3 II were tested by Western blot (N = 5 per group, **P<0.001 versus sham group, ##P<0.001, #P<0.05 versus MCAO group). (B) Expression of p-mTOR, STING, P62, Beclin1, LC3 I, and LC3 II were tested by Western blot (N = 5-6 per group, **P<0.01, ***P<0.001, *P<0.05 versus sham group, versus MCAO group). (C) Levels of interleukin (IL)-6, tumor necrosis factor-α, and IL-1β were measured by commercially available ELISA kits (N = 6 per group, **P<0.01 versus sham group, ##P<0.01, #P<0.05 versus MCAO group, P<0.05 versus MCAO+CMA group).

Figure 4

25-Hydroxycholesterol (25-HC) protected PC12 cells from I/R-induced cytotoxicity and inhibits autophagy following oxygen-glucose deprivation/reperfusion (OGD/R) in PC12 Cells. (A) PC12 cell viability is assessed by measuring CCK8. Results were shown as fold of control. (B–F) PC12 cells were pretreated with 25-HC and then cultured under OGD/R conditions or normoxia. Apoptosis analysis was performed, and representative flow cytometry images are shown. Quantifications of the percentage of apoptotic cells are shown (N = 3 per group, ***P<0.001, **P<0.01 versus sham group, ##P<0.01, #P<0.05 versus OGD/R group). (G) The expression of cleaved caspase3 was tested by immunocytofluorescent staining (N = 3 per group, ***P<0.001 versus sham group, ###P<0.001 versus OGD/R group). (H) The expressions of cleaved caspase-3, caspase-3, Bcl-2, and Bax were measured by Western blot (N = 3 per group, **P<0.01, *P<0.05 versus sham group, #P<0.05, versus OGD/R group).

Figure 4

25-Hydroxycholesterol (25-HC) protected PC12 cells from I/R-induced cytotoxicity and inhibits autophagy following oxygen-glucose deprivation/reperfusion (OGD/R) in PC12 Cells. (I) The expression of p-mTOR, STING, P62, Beclin1, LC3 I, and LC3 II were tested by Western blot (N = 3 per group, ***P<0.001, **P<0.01 versus sham group, ##P<0.001, ###P<0.001, ##P<0.01, #P<0.05 versus OGD/R group). (J) The protein levels of LC3 were detected by immunofluorescence (N = 3 per group, ***P<0.001 versus sham group, ###P<0.001 versus OGD/R group).

Figure 5

Involvement of the STING-mTOR/autophagy pathway in 25-Hydroxycholesterol (25-HC)-induced protection in PC12 cells. (A) The expressions of p-mTOR, STING, P62, Beclin1, LC3 I, and LC3 II were tested by Western blot (N = 3 per group, **P<0.01, *P<0.05 versus sham group, #P<0.05 versus OGD/R group, &P<0.05 versus OGD/R+CMA group). (B) The protein levels of LC3 were detected by immunofluorescence (N = 3 per group, **P<0.01 versus sham group, #P<0.05 versus OGD/R group, &P<0.05 versus OGD/R+CMA group).

Figure 6

Model describing the mechanism by which 25-Hydroxycholesterol protects against cerebral ischemiareperfusion injury. Left Figure: After cerebral ischemia and reperfusion, the expression of STING protein is activated and mTOR phosphorylation is inhibited, thereby increasing the expression of autophagy and achieving cerebral ischemia effect. Right image: After administration of 25-hydroxycholesterol, the expression of STING protein was inhibited, and mTOR phosphorylation was activated, thus inhibiting the expression of autophagy and achieving the effect of cerebral ischemia tolerance.