Abstract:

Objective To investigate the improvement effect of Panax notoginseng saponins （PNS） on neurological injury in the rats with ischemic stroke by regulating aerobic glycolysis， and to elucidate its mechanism. Methods Forty-eight rats were randomly divided into sham operation group， model group， PNS group （40 mg·kg^-1）， and PNS+phosphatidylinositol 3-kinase （PI3K） inhibitor LY294002 group （5 mg·kg^-1） PNS+LY294002 group， with 12 rats in each group. Except for sham operation group， the rats in other groups were subjected to middle cerebral artery occlusion/reperfusion （MCAO/R） method to establish ischemic stroke models. Longa scoring method was used to evaluate the neurological deficit of the rats in various groups. Triphenyltetrazolium chloride （TTC） staining method was used to detect the percentages of cerebral infarction area of the rats in various groups. HE staining and Nissl staining methods were used to observe the pathological changes and neuronal morphology in the brain tissue of the rats in various groups. Enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of lactate dehydrogenase A （LDHA）， interleukin （IL）-1β， and IL-18 in the brain tissue of the rats in various groups. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of hexokinase （HK）1， HK2， phosphofructokinase（PFK）1， PFK2， and pyruvate kinase M2 （PKM2） mRNA in the brain tissue of the rats in various groups. Immunofluorescence staining method was used to detect the levels of reactive oxygen species （ROS） in the cells and the perimeter/area （P/A） ratio of ionized calcium-binding adapter molecule 1 （Iba1）-positive microglia in the brain tissue of the rats in various groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling（TUNEL） method was used to detect the neuronal apoptosis rates in the brain tissue of the rats in various groups. Immunohistochemistry method and Western blotting method were used to detect the expression levels of PI3K/protein kinase B （Akt） pathway-related proteins and the ratios of phosphorylated PI3K （p-PI3K）/PI3K and phosphorylated Akt （p-Akt）/Akt in the brain tissue of the rats in various groups. Results Compared with sham operation group， the neurological deficit score of the rats in model group was significantly increased （P<0.05）， the percentage of cerebral infarction area was significantly increased （P<0.05）， and severe pathological damage was observed in the brain tissue of the rats； compared with model group， the neurological deficit scores of the rats in PNS group and PNS+LY294002 group were significantly decreased （P<0.05）， the percentages of cerebral infarction area were significantly decreased（P<0.05）， and the pathological damages in the brain tissue of the rats were alleviated； compared with PNS group， the neurological deficit score of the rats in PNS+LY294002 group was significantly increased （P<0.05）， the percentage of cerebral infarction area was significantly increased （P<0.05）， and the pathological damage in brain tissue was aggravated. The ELISA method results showed that compared with sham operation group， the levels of LDHA， IL-1β， and IL-18 in the brain tissue of the rats in model group were significantly increased （P<0.05）. Compared with model group， the levels of LDHA， IL-1β， and IL-18 in the brain tissue of the rats in PNS group and PNS+LY294002 group were significantly decreased （P<0.05）； compared with PNS group， the levels of LDHA， IL-1β， and IL-18 in the brain tissue of the rats in PNS+LY294002 group were significantly increased （P<0.05）. The results of RT-qPCR method showed that compared with sham operation group， the expression levels of HK1， HK2， PFK1， PFK2， and PKM2 mRNA in the brain tissue of the rats in model group were significantly decreased （P<0.05）. Compared with model group， the expression levels of the HK1， HK2， PFK1， PFK2， and PKM2 mRNA in the brain tissue of the rats in PNS group and PNS+LY294002 group were significantly increased （P<0.05）； compared with PNS group， the expression levels of the HK1， HK2， PFK1， PFK2， and PKM2 mRNA in the brain tissue of the rats in PNS+LY294002 group were significantly decreased （P<0.05）. The immunofluorescence staining results showed that compared with sham operation group， the level of ROS in cells in the brain tissue of the rats in model group was significantly increased （P<0.05）， and the P/A ratio of Iba1-positive microglia in the brain tissue of the rats was significantly decreased（P<0.05）； compared with model group， the levels of ROS in the cells in the brain tissue of the rats in PNS group and PNS+LY294002 group were significantly decreased （P<0.05）， and the P/A ratios of Iba1-positive microglia in the brain tissue of the rats were significantly increased （P<0.05）； compared with PNS group， the level of ROS in the cells in the brain tissue of the rats in PNS+LY294002 group was significantly increased （P<0.05）， and the P/A ratio of Iba1-positive microglia in the brain tissue of the rats was significantly decreased （P<0.05）. The TUNEL method results showed that compared with sham operation group， the neuronal apoptosis rate in the brain tissue of the rats in model group was significantly increased （P<0.05）； Compared with model group， the neuronal apoptosis rates in the brain tissue of the rats in PNS group and PNS+LY294002 group were significantly decreased （P<0.05）； compared with PNS group， the neuronal apoptosis rate in the brain tissue of the rats in PNS+LY294002 group was significantly increased （P<0.05）. The Western blotting method results showed that compared with sham operation group， the ratios of p-PI3K/PI3K and p-Akt/Akt in the brain tissue of the rats in model group were significantly decreased （P<0.05）； compared with model group， the ratios of p-PI3K/PI3K and p-Akt/Akt in the brain tissue of the rats in PNS group and PNS+LY294002 group were significantly increased （P<0.05）； compared with PNS group， the ratios of p-PI3K/PI3K and p-Akt/Akt in the brain tissue of the rats in PNS+LY294002 group were significantly decreased （P<0.05）. Conclusion PNS can effectively improve neurological deficit in the MCAO/R rats and reduce the level of ROS in brain tissue of the rats. Its mechanism may be related to activating the PI3K/Akt pathway， enhancing glycolytic capacity， and thereby promoting metabolic reprogramming of microglia.

Key words: Panax notoginseng saponins, Cerebral ischemia/reperfusion injury, Aerobic glycolysis, Apoptosis, Ischemia stroke