Lyophilized Powder of Catalpol and Puerarin Protects Neurovascular Unit from Stroke

Abstract

Hunting for an effective medicine for brain stroke has been a medical task in neuroscience for decades. The present research showed that the lyophilized Powder of Catalpol and Puerarin (C-P) in all the tested doses (65.4 mg/kg, 32.7 mg/kg, 16.4 mg/kg) significantly reduced the neurological deficiency, infarct volume and apoptotic cells in ischemic/reperfusion (I/R) rats. It also promoted astrocyte processes and prolonged neuron axons in infarct area. Further, it decreased MDA, NO, NF-κB/p65, TNF-α, IL-1β and IL-6 and enhanced the EPOR and GAF-43. 65.4 mg/kg and 32.7 mg/kg C-P could up-regulated EPO and VEGF significantly. In vitro, 49 μg/mL and 24.5 μg/mL C-P decreased the leakage of sodium fluorescein and increased the activity of γ-GTP. Additionally, it increased SOD and decreased MDA, NO, and LDH and decreased NF-κB/p65, TNF-α, IL-1β and IL-6 and unregulated EPO, EPOR, VEGF, and GAP-43. Only the dose of 49 μg/mL increased TEER and Claudin-5 and turned the typically damaged morphologies of neurons, astrocytes and endothelium into a favorable trend. These data imply that C-P improved the recovery of neurological deficiency in motor, sense, balance and reflex, and protected the whole NVU by anti-oxidative stress, anti-inflammation and up-regulating some protective factors. This research provides a candidate medicine for brain stroke and, at the same time, a pattern for drug study targeting NVU in vitro.

Lyophilized Powder of Catalpol and Puerarin Protected Cerebral Vessels from Ischemia by Its Anti-apoptosis on Endothelial Cells

“…The potential explanation for the difference is the different MCAO models, because neurological deficiency in MCAO rats generated by electrocoagulation was much weaker than it in MCAO rats prepared by nylon suture. Further, CP improved the outcome of cerebral ischemia including edema and infarct volume in MCAO mouse and ischemia/reperfusion rats 8, 9. In the present study, we further proved that CP increased the rCBF in the ischemic penumbral area.…”

“…Although we did not see significant improvement of neurological deficiency in CP-treated groups in the present study, we consider that CP was effective for neurological recovery as CP improved the neurological deficiency in our previous studies 8, 9. The potential explanation for the difference is the different MCAO models, because neurological deficiency in MCAO rats generated by electrocoagulation was much weaker than it in MCAO rats prepared by nylon suture.…”

“…Further, report shows that CP protected NVU in which vessel is a core element from apoptosis in vitro and in vitro 9. In addition to anti-apoptosis, CP improved cerebral circulation in the rat model of cerebral microcirculation disturbance induced by dextran infusion 29.…”

“…Mitochondria were isolated by kit for detecting Cyt-c. Western blot was repeated in 3 independent experiments with 6 wells of each group. Specific steps were followed the previous report 9. Respectively, primary antibodies were used in following concentrations: anti-HIF-1α :1500 Proteintech 12245), anti-cleaved caspase-3 (1:1000 CST 9662), anti-ERK (1:500 Proteintech 16443), anti-pErk (Thr202/Tyr204) (1:1000 CST 4370), anti-S6K (1:500 abcam ab36864), anti-pS6K (Thr389) (1:1000 CST9234), anti-P53 (1:1500 Proteintech 10442), anti-pP53 (Ser37) (Santa Cruz sc-9051), anti-pP53 (Ser15) (R&D AF1043), anti-AKT (1:1000, CST 9272), anti-pAKT (1:1000, CST 9271) p4EBP(1:1000, CST 2855), 4EBP(1:1000, CST 9955), VDAC (1:1000 CST 4866) and anti-β-actin (1:50000 Proteintech 60008).…”

“…The reason of choosing catalpol is that catalpol shows prospect in stroke 6 and depress 7 in pre-clinical research. Further, our previous studies show that the combination use of the two monomers well improved the outcome of middle cerebral artery occlusion (MCAO) mouse 8 and cerebral ischemia/reperfusion rats 9 in aspects of neurological deficiency, infarct volume, edema, inflammation, and oxidative stress. Besides, it increased cerebral CD31 positive cells in MCAO mouse 8 as well as protected human umbilical vascular endothelial cells (HUVECs) from oxygen glucose deprivation (OGD) 10, indicating that the protective effect of CP may be relative to vessel.…”