Abstract:

Objective To investigate the inhibitory effect of the combination of Astragaloside Ⅳ（AST Ⅳ）， Panax notoginseng saponins （PNS）， and borneol （APB） on neuroinflammation after cerebral ischemia/reperfusion （CI/R） in the rats by modulating aerobic glycolysis and microglial polarization， and to elucidate its synergistic mechanism. Methods Twenty-five healthy male SD rats were selected and randomly divided into sham operation group，model group， low dose of APB group， high dose of APB group， and positive control drug butylphthalide （NBP） group， with 5 rats in each group. The rat models of focal cerebral ischemia were established by middle cerebral artery occlusion （MCAO） using the suture occlusion method， while those in sham operation group did not undergo vascular occlusion.The rats in various groups received intragastric administration twice daily， starting at the onset of reperfusion after 2 h of ischemia. The rats in low dose of APB group were given 60 mg·kg^-1 borneol+80 mg·kg^-1 AST Ⅳ+200 mg·kg^-1 PNS， the rats in high dose of APB group were given 120 mg·kg^-1 borneol+160 mg·kg^-1 AST Ⅳ+ 400 mg·kg^-1 PNS， and the rats in NBP group were given 80 mg·kg^-1 NBP. The rats in sham operation group and model group were given equal volumes of pure water. At 2 h before execution， the modified neurological severity score（mNSS） of the rats in various groups were assessed. At 48 h after reperfusion， the rats were anesthetized， and the blood was collected from the abdominal aorta， and then the rats were sacrificed to collect the ischemic side of brain tissue. HE staining and Nissl staining were used to observe the neuronal damage and the number of Nissl bodies in the hippocampal region of brain tissue in the rats in various groups. Enzyme-linked immunosorbent assay （ELISA） method was used to detect the serum levels of interleukin-1β （IL-1β）， interleukin-10 （IL-10）， tumor necrosis factor-α （TNF-α）， and interleukin-4（IL-4） in the rats in various groups. Immunofluorescence staining was used to detect the polarization state of microglia towards M1 and M2 phenotypes in the brain tissue of the rats in various groups. Western blotting method was used to detect the expression levels of the key glycolytic rate-limiting enzymes lactate dehydrogenase A （LDHA）， glucose transporter 1 （GLUT1）， and hexokinase 2 （HK2） proteins in the brain tissue of the rats in various groups. Results ?According to the mNSS score， compared with sham operation group， the neurological deficit score of the rats in model group was significantly increased （P<0.05）； compared with model group， the neurological deficit scores of the rats in low dose of APB group， high dose of APB group， and NBP group were significantly decreased （P<0.05 or P<0.01）. The HE staining results showed regularly arranged neurons in the hippocampal region of the rats in sham operation group， and pyknosis and vacuolar degeneration of neuronal nuclei were observed in the rats in model group. Compared with model group， the neuronal damage was alleviated in low-dose APB group， high-dose APB group， and NBP group， as indicated by a reduction in pyknotic and vacuolated cells. Compared with sham operation group， the neuronal survival rate in the hippocampal region of the rats in model group was significantly decreased （P<0.05）； compared with model group， the neuronal survival rates in the hippocampal regions of the rats in low dose of APB group， high dose of APB group， and NBP group were significantly increased （P<0.05）. The Nissl staining results showed that compared with sham operation group， the number of Nissl bodies in neurons in the hippocampal region of the rats in model group was significantly decreased （P<0.05）； compared with model group， the numbers of Nissl bodies in the hippocampal neurons of the rats in low dose of APB group， high dose of APB group， and NBP group were significantly increased （P<0.05）. The ELISA results showed that compared with sham operation group， the levels of pro-inflammatory factors IL-1β and TNF-α in serum in the rats in model group were significantly increased （P<0.05）， while the levels of IL-10 and IL-4 in serum were significantly decreased （P<0.05 or P<0.01）； compared with model group， levels of IL-1β and TNF-α in serum in the rats in low dose of APB group， high dose of APB group， and NBP group were significantly decreased （P<0.05）， while the levels of IL-10 and IL-4 in serum were significantly increased （P<0.05 or P<0.01）. The immunofluorescence staining results showed that compared with sham operation group， the co-expression fluorescence intensity of inducible nitric oxide synthase（iNOS）/ionized calcium-binding adapter molecule 1（Iba-1） in the brain tissue of the rats in model group was significantly increased （P<0.01）， while that of arginase-1（Arg-1）/Iba-1 was significantly decreased （P<0.01）； compared with model group， the co-expression fluorescence intensities of iNOS/Iba-1 in the brain tissue of the rats in low dose of APB group， high dose of APB group， and NBP group were significantly decreased （P<0.05 or P<0.01）， while those of Arg-1/Iba-1 were significantly increased （P<0.05 or P<0.01）. The Western blotting results showed that compared with sham operation group， the expression levels of LDHA， GLUT1， and HK2 proteins in the brain tissue of the rats in model group were significantly increased （P<0.05）； compared with model group， the expression levels of LDHA protein in the brain tissue of the rats in low dose of APB group and high dose of APB group were significantly decreased （P<0.05）， while the expression levels of GLUT1 and HK2 proteins in high dose of APB group and NBP group were significantly decreased（P<0.05）. Conclusion ?APB can inhibit the polarization of microglia towards the M1 phenotype by downregulating the expression of glycolytic enzymes LDHA， GLUT1， and HK2 proteins， thereby alleviating inflammatory responses in brain tissue and improving neurological function in the rats after CI/R.

Key words: Cerebral ischemia/reperfusion injury, Glucose metabolism reprogramming, Inflammatory response, Astragaloside Ⅳ, Panax notoginseng saponins, Borneol