Rat microglia (RM, R1900) line was provided by Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. RM cells were cultivated in Microglia Medium (MM, Cat. No. 1901, Zhong Qiao Xin Zhou) at 37˚C in a 5% CO₂ incubator. The blank control group consisted of cells that received medium only. While a dose of 1 μg/mL lipopolysaccharide (LPS, S11060, Shanghai yuanye Bio-Technology Co., Ltd, China) was utilized as an active inflammation control. Then, cells were incubated with 1 μg/mL, 5 μg/mL or 10 μg/mL AX (B25542, Shanghai yuanye Bio-Technology Co., Ltd), respectively.

The eukaryotic vectors pcDNA3.1-PPAR-γ and its isotype control were established by Sangon, Shanghai, China. RM cells were inoculated in 12-well plates under a specific mass of 5.0 × 10⁴ cells/well after pre-transfection with 1 μg pcDNA3.1 plasmid for 24 h utilized Lipofectamine2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). Then, RM cells were digested 3 days after transfection.

PPAR-γ siRNA (si-PPAR-γ) and the scrambled siRNA (Scramble) were sourced from Shanghai GenaPharma Co., Ltd. Cells were transfected in term of the manufacturer’s instructions and harvested 24 h later for subsequent experiments.

Cell Counting Kit-8 (CCK-8) measurements (Sigma-Aldrich, St. Louis, MO, USA) were utilized to examine the RM cells viability. Briefly, cells were injected into 96-well microwells. After 24 h incubation, adding 10 µL of CCK-8 solubility product per well and incubating for 2 h with an additionally 5% CO₂ and 37˚C humidified. Precise measurement of 450 nm absorbance of each well by using a microplate reader.

The mature male Sprague-Dawley (SD) rats (weighted 260 ± 20 g) utilized in this research were acquired from the Animal Center of Xi’an Jiaotong University, and entire animal agreements were managed in terms of the “Guidelines for the Care and Use of Laboratory Animals”, and the animal trials were confirmed by the Shaanxi Provincial People’s Hospital Animal Research Committee. I/R injury was performed by middle cerebral artery occlusion/reperfusion (MCAO/R) as antecedently reported [17]. Briefly, rat cerebral ischemia was operated by 60-min invertible MCAO upon isoflurane anesthesia. An automatic temperature control feedback system was utilized to sustain anal temperature at 36.5˚C ± 0.5˚C during surgery. Unilateral MCAO was executed by inserting a 6.0 mm monofilament (Doccol, Redlands, CA) through the external carotid stump into the right internal carotid artery 6 mm from the internal carotid/pterygopalatine bifurcation using a midline abdominal neck incision. 60 min occlusion followed by thread withdrawal for reperfusion. Fifty rats were casually separated into five groups: sham operation group (Sham), MCAO/R group, MCAO/R+AX group, MCAO/R+AX+ Scramble group and MCAO/R+AX+ si-PPAR-γ group, 10 per group. Sham group rats were subjected to the uniform surgical step, just the stitching was not got into the inner carotid artery. MCAO/R+AX group rats were subcutaneously injected with 10 mg/kg AX daily for 7 days before operation. MCAO/R+AX+ Scramble group or MCAO/R+AX+ si-PPAR-γ group rats were respectively injected with 10 μL of scrambled siRNA or PPAR-γ siRNA in the ventricle daily for 3 days before surgery. All of the rats were slaughtered at 7 d of reperfusion. The measurement of the MCAO/R-stimulated infarct was determined by triphenyl tetrazolium chloride (TTC) staining.

After MCAO for 7 d, rat brain tissue was shifted and cut into 71-mm coronal slices, which were placed at 37˚C and dipped in a 2% 2,3,5-triphenyl-2H-tetrazolium chloride fluid for 15 min. At this point, normal brain tissues were dyed red, while the infarct tissues were dyed light gray. Subsequently, the sections were taken pictures and evaluated using Image-Pro Plus 6.0 (Media Cybernetics), and the infarct volume (percent) of the sections was computed after modification for dropsy.

All animals were tested for foot failure and a modified neurological severity score (mNSS) was procured at 1, 3, 5 and 7 days after MCAO/R operation. mNSS is a complex which utilized to appraise neurological characteristic grounded on motion, sensation, equilibrium, and reflex measuring on a range of 0 to 18 (normal score, 0; maximum deficit score, 18), and higher marks were intended to biggish nerve damage.

Trizol reactant (Invitrogen) was utilized to purify entire RNA from RM cells or brain tissues in terms of the manufacturer’s explanations, NanoDrop ND-1000 (NanoDrop Technologies) was utilized to confirm the density of depurative RNA specimens. SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) was utilized for reverse transcription of RNA to cDNA. Additionally, real-time quantitative PCR experiments were performed with the Platinum^TM Taq DNA Polymerase (Thermo Fisher Scientific) with SmartChip Real-Time PCR System (TaKaRa Bio, Dalian, China). Besides, the PCR response system involved 1.0 μL of RT primer, 1 μL of cDNA sample, 12.5 μL of Taq DNA Polymerase, and double distilled H₂O was utilized to complement the remaining volume, and the primer sequences utilized in this research were as below: Cluster of differentiation 206 (CD206, sense: 5’-CTT CGG GCC TTT GGA ATA AT-3’, antisense: 5’-TAG AAG AGC CCT TGG GTT GA-3’); Cluster of differentiation 68 (CD68, sense: 5’-GCT ACT GGC AGC CCC AGG G-3’, antisense: 5’-GCT CTT GGT AGT CCT GTG G-3’); inducible nitric oxide synthase (iNOS, sense: 5’-CCC AGA GTT CCA GCT TCT GG-3’, antisense: 5’-CCA AGC CCC TCA CCA TTA TCT-3’); GADPH (sense: 5’-GAG TCA ACG GAT TTG GTC GT-3’, antisense: 5’-AGC ACT GTG TTG GCG TAC AG-3’). Genes expression levels were standardized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the 2^−ΔΔCt manner.

RM cells and rats brain tissues were dissolved with RIPA lysis buffer (Elabscience Biotechnology, Wuhan, China). BCA protein assay kit (Elabscience Biotechnology) was utilized to determine the protein density. Afterwards, equivalent quantities of proteins (30 μg/lane) were isolated by 10% SDS-PAGE and electro-transferred onto 0.45 μm PVDF membranes (E-BC-R266, Elabscience Biotechnology). Subsequently, membranes were immerged in 5% non-fat milk for 2 h in an indoor environment, and then primary antibodies provided from Abcam comprising rabbit monoclonal anti-PPAR-γ (1:1000, ab272718), anti-phosphorylated P65 (p-P65, 1:1000, ab76302) and anti-P65 (1:2000, ab76311) and mouse monoclonal anti-GADPH (1:2000, ab9485) were respectively appended to petri dish co-cultivating with membranes overnight at 4˚C. Later, secondary antibody horseradish peroxidase (HRP)-tagged rabbit anti-mouse IgG (1:1000, #58802, Cell Signaling Technology, Boston, MA, USA) was affixed to membranes at indoor temperature for 2 h. GADPH was selected as the internal reference protein. In addition, proteins expression levels were determined by chemiluminescence, and ImageJ software (National Institutes of Health, Bethesda, MA, USA) was utilized to quantify proteins expression levels.

The excretion of cytokines containing IL-1β, IL-6 and TNF-α in RM cells and rats brain tissues were tested by specific ELISA kits (TaKaRa Biotechnology) pursuant corresponding manufacturer’s instructions.

The experimental data were determined by SPSS 22.0 (SPSS Inc., Chicago, IL, USA) and appeared as mean ± SEM. While P < 0.05 was recognized as statistical significance. The diversity between two unpaired instances was assessed by student’s t test. The diversity among more than three groups was assessed by one-way ANOVA with Tukey’s post hoc test or by two-way ANOVA with Bonferroni post hoc test.

To study the effect of AX on microglia activization, RM cells were first co-cultivated with diverse concentration of AX. As shown in Figure 1(A), AX at doses

ranging from 1 to 40 µg/mL were noneffective in cell viability. Compared with the control group, LPS induction increased the levels of CD68 (Figure 1(B)) and iNOS (Figure 1(C)) mRNA and the secretion of cytokines IL-1β, IL-6 and TNF-α (Figures 1(E)-(G)), and decreased the expression of CD206 mRNA (Figure 1(D)) and PPAR-γ protein (Figure 1(H)) levels. Compared with the LPS stimulation group, AX treatment decreased the levels of CD68 (Figure 1(B)) and iNOS (Figure 1(C)) mRNA and the secretion of cytokines IL-1β, IL-6 and TNF-α (Figures 1(E)-(G)), and increased the expression of CD206 mRNA (Figure 1(D)) and PPAR-γ protein (Figure 1(H)) levels. While the effect of high-dose AX (10 µg/mL) treatment group was superior than that of medium- (5 µg/mL AX) and low-dose (1 µg/mL AX) groups.

To explore the influence of PPAR-γ protein on microglia activation, LPS-induced RM cells were co-cultivated with pcDNA-PPAR-γ. After that, the cell functions were determined. The results displayed that compared with the LPS+ Vector group, the expression of CD68 (Figure 2(B)) and iNOS (Figure 2(C)) mRNA levels and the secretion of cytokines IL-1β, IL-6 and TNF-α (Figures 2(E)-(G)) were enhanced, and the levels of PPAR-γ protein (Figure 2(A)) and CD206 mRNA (Figure 2(D)) were decreased in pcDNA-PPAR-γ treatment group.

To further probe the regulatory mechanism of AX on microglial activation, LPS-induced RM cells were incubated with 10 µg/mL AX after transfection with si-PPAR-γ. Western blotting analysis displayed that compared with the control group, the level of PPAR-γ protein (Figure 3(A)) was decreased and the level of p-P65 protein (Figure 3(B)) was increased after LPS induction. Compared with the LPS induction group, PPAR-γ protein level was increased and p-P65 protein was decreased after AX treatment. While PPAR-γ interference downregulated the level of PPAR-γ protein and upregulated the level of p-P65 protein. As determined by RT-qPCR and ELISA assays, PPAR-γ interference increased the levels of CD68 (Figure 3(C)) and iNOS (Figure 3(D)) mRNA and the secretion of cytokines IL-1β, IL-6 and TNF-α (Figures 3(F)-(H)), and decreased the expression of CD206 mRNA (Figure 3(E)). All these findings suggested that the inhibitory effect of AX on LPS-induced microglial activation might be related to the PPAR-γ/NF-κB pathway.

Similarly, compared with the LPS+AX group, PPAR-γ antagonist GW9662 treatment increased the expression of p-P65 protein level (Figure 4(A)), CD68 (Figure 4(B)) and iNOS (Figure 1(C)) mRNA levels and the secretion of cytokines

IL-1β, IL-6 and TNF-α (Figures 4(F)-(H)), and decreased the levels of PPAR-γ protein (Figure 4(A)) and CD206 mRNA (Figure 4(E)).

In order to investigate the effect of AX on rat cerebral I/R injury, we firstly

established a rat MCAO/R model. The results revealed that compared with the sham operation group, the neurological damage of the rats in the MCAO/R model group was significantly severe (Figure 5(A)), and the levels of CD68 (Figure 5(B)) and iNOS (Figure 5(C)) mRNA and the secretion of cytokines

IL-1β, IL-6 and TNF-α (Figures 5(E)-(G)) in rats brain tissues were distinctly enhanced. Compared with the MCAO/R model group, AX treatment prominently ameliorated the neurological damage of rats (Figure 5(A)), and dramatically decreased the expression of CD68 (Figure 5(B)) and iNOS (Figure 5(C))

mRNA and the secretion of cytokines IL-1β, IL-6 and TNF-α (Figures 5(E)-(G)) in rats brain tissues, and dramatically reduced the expression of CD206 mRNA (Figure 5(D)). These results suggested that AX protects rats from I/R-induced nerve injury.

To investigate the pathogenesis of rat cerebral I/R injury, MCAO/R model rats were treated with AX with or without injection si-PPAR-γ. Western blotting results displayed that compared with Sham group, the level of PPAR-γ protein (Figure 6(A) & Figure 6(B)) & was prominently reduced and the level of p-P65 protein (Figure 6(A) & Figure 6(C)) was prominently increased in the MCAO/R model group rats. Compared with MCAO/R model group, AX treatment increased the level of PPAR-γ protein and decreased the level of p-P65 protein in rats brain tissues. While si-PPAR-γ injection reduced the level of PPAR-γ protein and enhanced the level of p-P65 protein. TTC staining results demonstrated that compared with Sham group, the rat brain infarct volume (Figure 6(C)) was prominently enhanced in MCAO/R model group, and AX treatment observably mitigated rat brain infarct volume, while si-PPAR-γ injection reversed the effect of AX. As determined by RT-qPCR and ELISA assays, compared with the AX treatment group, si-PPAR-γ injection markedly increased the expression of CD68 (Figure 6(D)) and iNOS (Figure 6(E)) mRNA and the excretion of cytokines IL-1β, IL-6 and TNF-α (Figures 6(G)-(I)) in rats brain tissues, and dramatically reduced the expression of CD206 mRNA (Figure 6(F)). The above results suggested that AX alleviates nerve injury after I/R in rats by regulating the PPAR-γ/NF-κB pathway.