Fig 2: mRNA levels of (A) nNOS and (B) iNOS determined by reverse-transcription quantitative polymerase chain reaction with ß-actin used as an internal standard. The mRNA levels of nNOS and iNOS were significantly increased at post-operative day 10 when compared with those of the controls. The increase of iNOS continued until post-operative day 14, while that of nNOS did not. Values are expressed as the mean ± standard deviation. *P<0.05 vs. sham group at the same time-point, #P<0.05 vs. the same group of mice at day 7. iNOS, inducible nitric oxide synthase; nNOS, neuronal NOS; S, sham; T, tumor.

Fig 3: Immunocytochemical localization and expression of nNOS and iNOS in the spinal cord of tumor-bearing mice. (A) nNOS immunostaining in the lumbar spinal cord at day 10 after tumor-cell inoculation. (C) iNOS immunostaining in the lumbar spinal cord at day 14 after tumor-cell inoculation. Magnification, x40. The nNOS and iNOS immunoreactive cells (arrows) were mainly located in the dorsal horn of the spinal cord (laminae I-II) and a few in the ventral parts and around the central canal. (B) nNOS and (D) iNOS immunostaining in the IPSI side of the superficial dorsal horn at higher magnification, ×200. Statistical analysis of mean optical density of (E) nNOS and (F) iNOS. Values are expressed as the mean ± standard deviation. *P<0.05 vs. sham group at the same time-point, #P<0.05 vs. the same group of mice at day 7. IPSI, ipsilateral; iNOS, inducible nitric oxide synthase; nNOS, neuronal NOS.

Fig 4: Effect of safflor yellow B (SYB) on target protein expression in RAECsRAECs were used to establish OGD model. Total proteins in RAECs from different groups were extracted and measured. Thirty micrograms of total proteins were loaded per lane, and separated by SDS-PAGE and transferred to PVDF membrane. After incubation with secondary antibodies, the expression levels of HIF-1a, S100A1, p-iNOS, iNOS, p-eNOS, eNOS, caspase3,Bax, Bcl-2 and ß-actin were visualized using chemiluminescence method. A represent Western blots in panel (1, 2, 3, 4, 5, 6, 7, 8 represent control group, OGD group, SYB group, antimiR-199a group, antimiR-199a+SYB group, L-NAME group, L-NAME+SYB group and antimiR-199a+L-NAME+SYB group respectively). (A1, A2, A3, A4, A5, A6) represent the relative ratio of p-iNOS/iNOS and p-eNOS/eNOS, HIF-1a, S100A1,relative ratio of Bax/Bcl-2, caspase3 respectively. Data were presented as mean ± S.D. (n = 3). One-way ANOVA test was used to determine statistical significance. *P < 0.05 or**P < 0.01 vs. control group, #P < 0.05 or ##P < 0.01 vs. OGD group, ?P < 0.05 or D?P < 0.01 vs. SYB+antimiR-199a, +P< 0.05 or ++P < 0.01 vs. L-NAME+SYB.

Fig 5: Effect of safflor yellow B (SYB) on the activities of protein kinase C (PKC), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS)(A1, A2, A3) Effect of safflor yellow B (SYB) on the activities of PKC, iNOS and eNOS in vitro. RAECs were used to establish OGD model. RAECs were homogenized and centrifuged at 4°C, and the supernatants were used to measure the activities of PKC, iNOS and eNOS according to the procedure described as assay Kits. A1, A2, A3 represent the activities of PKC, iNOS and eNOS in RAECs. (B1, B2, B3) Effect of safflor yellow B (SYB) on the activities of PKC, iNOS and eNOS in vivo. Rats were divided into three groups: normal, hypoxia, SYB. Except for normal group. all rats in other groups were reared in hypoxic cabin. The serum and vascular endotheliums were obtained aftet 4 weeks. B1 represents PKC activity in vascular endotheliums, and B2 and B3 represent the activities of iNOS and eNOS in serums. Data were presented as mean ± S.D. (n = 8 in tissues or serums, n = 3 in cells). One-way ANOVA test was used to determine statistical significance. **P < 0.01 vs. normal group or control group, #P < 0.05 or ##P < 0.01 vs. hypoxia group or OGD group, D?P < 0.01 vs. SYB+ antimiR-199a, +P< 0.05 or ++P < 0.01 vs. L-NAME+SYB.