Animals and study design
The animals are purchased from the company (Jihui Experimental Animal Breeding Co., Ltd, Shanghai, China). A total of 100 male SD rats ranging from 200 g to 250g were selected. Pentobarbital 30mg/kg was injected intraperitoneally and the induction time was 20min. In this study, grouped as follows :(1) Sham operation group; (2) Bilateral common carotid artery ligation (BCCAO) was performed in the experimental group, and data collection was prevented if the animals died prematurely. (3) SCFAs (acetate: propionate: butyrate at a ratio of 3: 1: 1, 500mg/kg, intragastrically, for 7days before BCCAO) +BCCAO. The rats were fed under normal oxygen condition for 1, 3 and 7 days before death. Animals were killed for cervical dislocation. Animal treatment and experiments shall be approved by the Shanghai Municipal Commission for Animal Protection and Use.
To observe the effect of SCFAs on astrocyte activation via SGK1 (lentivirus transfection,caudal vein). The rats were divided into sham operation group; BCCAO group; BCCAO+SCFAs group; BCCAO+SCFAs+overexpression SGK1 group; BCCAO+SCFAs+si-SGK1 group; SCFAs group. The rats were fed under normal oxygen condition for 1 day and then killed.
Primary Culture of Oligodendrocytes
Concrete details were in reference . To explore the IL-6 on oligodendrocytes. Grouped as follows: the control group; 20ng/ml IL-6 group; 20ng/ml IL-6 + 20ng/ml IL-6Ra (Sarilumab, anti-IL-6Rα) group; 20ng/ml IL-6Ra group.
Primary Cultured Astrocytes
Briefly, the mixed cells were isolated from 1 days old SD rats. The mixed culture method was shown in literature . Mixed cells for 10 d, and then oscillated at 180 RPM and 36.5℃ for 1h to remove OPCs and microglias. Standing under normal conditions for 24h, the cells were treated differently. In this study, astrocytes with a purity of more than 90% were cultured.
Intervention of Astrocytes
To see if SCFAs had an effect on NLRP3 ,IL-6, CCL2, and IP-10 in astrocytes, the astrocytes were grouped as follows: the control group; oxygen glucose deprivation (OGD) group; OGD+20ng/ml SCFAs group; SCFAs group.
To examine whether SCFAs affect SGK1 in astrocytes. Grouped as follows: the control group; OGD group; OGD+SCFAs group; OGD+SCFAs+SGK1 group; OGD+SCFAs+si-SGK1 group (The transfection method was shown in Ref. ).
To examine whether SCFAs affect IL-6 via SGK1 signaling pathway. Grouped as follows: the control group; OGD group; OGD+SCFAs group; OGD+SCFAs+SGK1 group; OGD+SCFAs+si-SGK1 group; SCFAs group.
IL-6 protein levels were determined by western blotting, concrete details were in reference . The primary antibodies used were as follows: IL-6 (1:1,000,Bosterm,China), NLRP3 (1:1,000, CST; USA), IL-6 receptor (1:1,000, Santa Cruz, USA), Bax (1:1,000, Santa Cruz,USA); Bcl-2 (1:1,000), GFAP (1:1,000), CCL2 (1:1,000), IP-10 (1:1,000) β-actin (1:1,000) [all from Cell Signaling Technology]. After washing with TBST for three times, the cell membrane was treated with HRP were incubated for 1 h. Then immunoluminescence was performed.
Each group was incubated with the following antibodies, against IL-6 (1:100) and GFAP (1:100). anti-IL-6R (1:100). To verify the apoptosis and proliferation of OPCs: anti-cleaved caspase-3 (1:100), anti-BrdU (1:100, concrete details were in reference ), and anti-NG2 or anti-PDGFR-α (1:100) were utilized. 4°C overnight and then incubate with secondary antibody and observe the slice under the fluoroscope.
sections were gavaged in 0.1M phosphate buffer with a mixture of 2% paraformaldehyde and 3% glutaraldehyde. then, the brain was removed and coronal sections (about 1mm thick) were cut. They were then fixed in 1% osmium tetroxide for 2 hours, dehydrated, and subsequent processing. The ultrathin sections were cut and observed on electron microscope (FEI Corporation, Hillsboro, OR).
All data were evaluated using SPSS13.0. Distribution values are expressed as mean ± standard deviation. Four groups of univariate data were analyzed by univariate analysis of variance under homogeneity of variance. In addition, Welch analysis of variance was used for analysis. If the data were homogenous of variance, the least significant difference (LSD) method was used for multiple comparisons. P<0.05 was statistically significant.