Effect of Jianpi Bushen Huoxue Prescription on Rat Brain Microvascular Endothelial Cells Based on HIF-1α/VEGF Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo observe the effects of Jianpi Bushen Huoxue prescription （JPBSHX） on rat brain microvascular endothelial cells （RBMECs） based on hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF） signaling pathway， aiming to provide a theoretical basis for the treatment of ischemic stroke. MethodTwelve 8-week-old male SPF-grade SD rats were selected. Eight of them were randomly chosen and given 3.25 g·mL-1 JPBSHX solution by gavage at a dose of 10 mL·kg-1 for 5 consecutive days to prepare the medicated serum， which was then preserved for later use. The remaining four rats were given the same volume of normal saline. Follow-up operations were the same as those of the above eight rats. Normal rat serum was collected and stored for later use. RBMECs were revived， cultured， passaged， and randomly divided into five groups： normal group （20% normal rat serum+80% high glucose DMEM）， model group （hypoxia-reoxygenation injury） （20% normal rat serum+80% glucose-free DMEM）， medicated serum group （20% JPBSHX-medicated serum+80% glucose-free DMEM）， medicated serum+HIF-1α inhibitor group （20% JPBSHX-medicated serum+HIF-1α inhibitor 1 mg +80% glucose-free DMEM）， and medicated serum+VEGF inhibitor group （20% JPBSHX-medicated serum +VEGF inhibitor 1 mg+80% glucose-free DMEM）. The relative protein expression levels of Claudin-1 and Claudin-5 in RBMECs， the expression levels of HIF-1α and VEGF in RBMEC culture supernatants， the repair ability of RBMECs， and the number of nodes， microvessels， and their lengths after 72 h of culture were observed in each group. ResultAfter 24 h of reoxygenation， the scratch healing rate in the model group was significantly lower than in the normal group （P<0.01）. Compared with the result in the model group， the scratch healing rates significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group （P<0.05）. However， the healing rates in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group were significantly lower than that in the medicated serum group （P<0.05）. The number of nodes， microvessels， and total length of microvessels in the model group were significantly lower than those in the normal group （P<0.01）. These indicators significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group compared with those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group compared with those in medicated serum group （P<0.05）. The relative expression levels of Claudin-1 and Claudin-5 proteins were significantly lower in the model group than in the normal group （P<0.01）. These levels were significantly higher in medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. The expression levels of HIF-1α and VEGF in the RBMEC culture supernatants were significantly lower in the model group than those in the normal group （P<0.01）. These levels were significantly higher in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. ConclusionJPBSHX can promote the proliferation， migration， and angiogenesis， such as tubule formation， of RBMECs damaged by hypoxia-reoxygenation injury， and this effect may be achieved through the regulation of the HIF-1α/VEGF signaling pathway.