This project was all approved by the Ethics Committee of Yiyang Central Hospital (Hunan Province, P. R. China). Reference number: H201803043. Written informed consent was provided before sample collection. All animal experiments were approved by the Research Center for Medicine & Department of Laboratory Animal Science of Nanhua University. Reference number: 201506164.

Thirty-six CD-1 mice (25-30 g, males) were purchased from the Research Center for Medicine & Department of Laboratory Animal Science of Nanhua University. Mice were randomly divided into the control group (n = 12), the sham group (n = 12), and the MCAO group (n = 12). In the MCAO group, 3% chloral hydrate (300 mg/kg, i.p.) was used to anesthetize the mice. Then a silicon-coated monofilament nylon suture was used to occlude the origin of MCA. B-ultrasound was used to detected mice's cerebral blood flow, and the successful MCAO modeling was confirmed by the reduction of hemispheres' blood flow. The sham group underwent the same anesthesia and surgical procedure except for the suture ligation of MCA. Mice in the control group did not receive any surgical operations. 40 μL vein blood was collected from the tail of each mouse at different times after modeling.

HBMECs were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 U/mL penicillin-streptomycin, and grew in an incubator in 37 °C with 5% CO₂. The culture was replaced every other day. OGD treatment was performed by culturing cells in DMEM containing no glucose (Gibco, USA), and incubating cells in an incubator (Thermo Scientific, USA) with 1% O2, 5% CO2, and 94% N2 for different times (Zhou et al., 2019).

Negative control (NC) and three small interfering RNAs targeting ROR (si-ROR) were purchased from Genepharm. Co. (Shanghai, P. R. China). The sequences of siRNAs were provided in Supplementary Table 1. Lipofectamine 3000 (Invitrogen, USA) was used to perform the transfections. Briefly, the original culture was removed and replaced with a culture containing a mixture of siRNAs and Lipofectamine 3000 (1 : 1). After 6 h of incubation, culture was replaced with normal medium containing no FBS. After 48 h of transfection, ROR expression was detected. The silencing efficiency was evaluated by the down-regulation of ROR at 48 h post-transfection. In this study, si-ROR-3 was finally used to silence ROR expression.

Cells were lysed on ice and centrifuged at 12,000 g for 5 min to separate cell debris. Total protein concentration was detected by the BCA method (BCA Protein Assay Kit, Beyotime, P. R. China). Then equal amount of proteins from each group were separated on 10% SDS-PAGE gels and transferred onto a polyvinyl difluoride (PVDF) membrane. After incubated with 5% BSA (Bovine Serum Albumin) at 37 °C for 1 h, the membrane was incubated with one of the following antibodies overnight at 4 °C: anti-Bax antibody (ab32503, Abcam), anti-ASK-1 antibody (8662, Cell Signaling Technology), anti-phospho-ASK-1 antibody (3764, Cell Signaling Technology), anti-STRAP antibody (AP29336; One World Lab), anti-14-3-3 antibody (9636; Cell Signaling Technology), anti-caspase 8 antibody (ab25901, Abcam), anti-TNF-α antibody (ab1793, Abcam), and anti-tubulin antibody (ab6160, Abcam). Then the membrane was incubated with secondary antibody (ab6721 or ab6789, Abcam) for 1 h at 37 °C. Finally, the membrane was photographed by ECL Imaging System (Tanon 6060, USA).

Annexin V fluorescein isothiocyanate (FITC)/apoptosis detection kit (Sigma, USA) was used to evaluate cell apoptosis according to the manufacturers' instructions. Briefly, 5 μL annexin V-FITC and 10 μL propidium iodide were added to cell suspensions and incubated for 15 min at room temperature. Then stained cells were analyzed by BD FACS Calibur™ flow cytometer (BD Biosciences, USA).

Cells were washed with cold phosphate buffered saline (PBS) twice and fixed with 4% polyoxymethylene for 10 min at room temperature. Then cells were incubated with 5% BSA for 0.5 h at 37 °C. After that, anti-cleaved caspase-3 antibody (1 : 200; Cell Signaling Technology, USA) was added and incubated with cells overnight at 4 ℃. Followed by incubating with secondary antibody (BA1105, BOSTER) for another 1 h at 37 °C. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was added and incubated for another 2 min to indicate the nucleus. Finally, cells were washed twice using PBS and were observed under an inverted microscope. (Olympus, Japan).

Lysates were generated from the brain tissues of ICR mice. The lysates were precleared with Protein G beads in advance. After that, the precleared lysates were incubated with 5 μg/mL of anti-ASK-1 antibody (8662; Cell Signaling Technology), or anti-P-ASK-1 antibody (28201-1-AP, protein tech group) to pull down the target complexes. Normal rabbit IgG (5 μg/mL) was also added as a negative control. All samples were incubated overnight using a constant rotation at 4 ℃. Then protein G beads were added and incubated for another 2 h. After washed 6 times by 1 × lysis buffer, protein G beads were boiled in 1 × loading buffer, and the co-precipitated complexes were then separated on SDS-PAGE and used for western blotting analysis.

Two hundred and twenty-three patients (145 men and 78 women, mean age: 67.76±6.32) diagnosed as IS between December 2015 and April 2018 at Yiyang Central Hospital, Hunan, P. R. China, were recruited in this study. Patients were classified into three groups: large-artery atherosclerosis (LAA), cardioembolism (CE), and small-vessel occlusion (SVO) according to the results of magnetic resonance imaging and computed X-ray tomography. At admission, the National Institutes of Health Stroke Scale (NIHSS) score, the infarct volume, and the high sensitivity C-reactive protein (Hs-CRP) were all assessed. The modified Rankin Scale (mRS) of subjects was recorded 6 months after hospitalization. One hundred fifty-five controls (104 men and 51 women, mean age: 65.53±7.98) from the health examination center were also collected. For IS patients, blood samples were collected at different times after symptom onset (within 3-12 [n = 64], 12-24 [n = 116], 24-36 [n = 32], 36-48 [n = 11]). For healthy controls, blood samples were all obtained at the time of physical examination.

Total RNA was extracted by Invitrogen™ TRIzol™ Reagent (Thermo Fisher Scientific, USA). The concentration of total RNA was detected by Nanodrop 2000 (Thermo, USA). RNA was reverse transcribed into cDNA using PrimeScriptTM RT Master Mix (Takara, Japan). ROR expression was detected on the Bio-Rad CFX96 (Inc., Hercules, CA, USA) using SYBR Green qPCR Mix (CWBIO, P. R. China). The sequence of primers was provided in Supplementary Table 1. Two control transcripts, β-actin, and GAPDH were both detected in this study. Compared to β-actin, the intra- and inter-group variabilities of GAPDH expression were smaller, so GAPDH was finally chosen as the control. The expression of ROR was normalized to GAPDH. All experiments were in triplicates. ROR level was calculated using the 2-ΔCT method, where ΔCT = CT(target) - CT(reference).

SPSS version 20.0 was used to perform statistical analyses. In this study, normally distributed data were presented as mean±standard deviation (M±SD), while skewed data were presented as median (interquartile range, IQR). For normally distributed variables, Student’s t-test was used to evaluate the difference between the two groups, and one-way ANOVA was used for the analysis of multiple groups. Categorical variables were analyzed by the χ² test. Correlations were analyzed using Pearson or Spearman correlation analysis. In respect to animal experiments and human study, the group information as defined by numbers and staff who were responsible for data collection and data analysis were all blind to the status of subjects. In this study, P < 0.05 was considered to be statistically significant.

Fig. 1A shows mice brain images after MCAO operation. Brain tissue in the MACO group showed a significant pale region caused by ischemia. Results showed that ROR expression in mice from the MCAO group was markedly increased compared to that in the sham group and the control group, while there’s no significant difference between ROR level in mice from the control group and the sham group (Fig. 1B). The expression of ROR after MACO establishment rapidly raised in the first 2 h, then kept stable within 2 to 48 h, and shows a slow-upward trend after 48 h (Fig. 1C).

Figure 1.

The expression pattern of ROR in MCAO mice. (A). Mouse brain tissue after MCAO establishment. (B). ROR level in the MCAO group was up-regulated significantly compared to that in the sham group and the control group at 24 h after the IS model establishment. (C). ROR expression pattern during IS model establishment. Data are shown as mean±SEM, **P < 0.001.

We further detected the expression levels of some apoptosis-related proteins. As the results showed, expressions levels of Bax and cleaved caspase-3 significantly increased after OGD treatment, as well as the apoptosis rate of HBMECs, while si-ROR amplified these effects (Fig. 2A, B, C). Besides, ROR expression in HBMECs could also be up-regulated by OGD treatment, whose expression pattern was similar to that in MACO mice: increased rapidly in the first 3 h, then remained stable and gradually increased within 3 to 48 h (Fig. 2D).

Figure 2.

Apoptosis of HBMECs in different groups. (A). Results of flow cytometry showed that apoptosis of HBMECs was significantly up-regulated in response to si-ROR treatment. (B). Results of western blot showed that OGD significantly up-regulated Bax expression, and this effect can be amplified by si-ROR. (C). Results of immunofluorescence showed that OGD significantly up-regulated cleaved caspase-3 expression, and this effect can be amplified by si-ROR. (D).The expression pattern of ROR after OGD treatment. Data are shown as mean±SEM, **P < 0.001.

We further analyzed the apoptosis complexes in different groups. The results showed that OGD treatment could significantly promote the combination of STRAP and 14-3-3 to ASK-1, thus inhibited the interaction of TNF-α and P-ASK-1. In other words, OGD treatment to HBMECs promoted ASK-1/STRAP/14-3-3 complex formation and inhibited TNF-α/P-ASK-1 complex formation, while si-ROR amplified these effects (Fig. 3A, B).

Figure 3.

Apoptosis complexes of HBMECs in different groups. (A. C. D. E). OGD treatment significantly reduced ASK-1/STRAP/14-3-3 complex formation, and si-ROR amplified these effects. (B. F) OGD treatment enhanced the formation of TNF-α/P-ASK-1 complexes, and si-ROR amplified these effects. Data are shown as mean±SEM, *P < 0.05, #P < 0.001.

ROR level in IS patients went to the hospital at different times after IS onset was analyzed, and no significant difference was observed (P > 0.05, Fig. 4A). However, we did observe that IS patients had significantly higher ROR expressions compared to controls (P < 0.001, Fig. 4B), which is consistent with the results from the animal study and cell study. Further research showed that patients in the LAA group had the highest ROR expressions compared with patients in CE, SVO, and control groups, while there’s no significant difference in ROR expressions between CE and SVO group (Fig. 4C).

Figure 4.

Serum ROR expression in different groups. (A). No significant difference was found in ROR expression among patients admitted to hospital at different times. (B). Compared to controls, ROR expression was up-regulated significantly in IS patients. (C). ROR expression in large-artery atherosclerosis (LAA) was up-regulated most significantly. Data are shown as mean±SEM, *P < 0.05, **P < 0.001.

To evaluate the value of ROR in reflecting disease severity of IS, we further analyzed the correlation between serum ROR expression level and the clinical characteristics of IS patients. As the results showed, ROR expression level was negatively correlated with NIHSS scores (r = -0.708, P < 0.001, Fig. 5A), infarct volume (r = -0.579, P < 0.001, Fig. 5B), and Hs-CRP level (r = -0.683, P < 0.001, Fig. 5C). Also, there was a significant negative correlation between serum ROR expression level and mRS score (r = -0.728, P < 0.001, Fig. 5D).

Figure 5.

Relationship between serum ROR level and the severity of IS. (A) Serum ROR level had a positive correlation with the National Institutes of Health Stroke Scale (NIHSS) score of IS patients. (B) Serum ROR level had a positive correlation with the infarct volume. (C) Serum ROR level had a positive correlation with serum Hs-CRP. (D) Serum ROR level had a positive correlation with the modified Rankin Scale (mRS) score.