Antiischemia-reperfusion Injury In Neuronal Cells And Antineuroinflammation Effect Of Polygalasaponin F In Vitro

Ischemic cerebrovascular disease is the most common clinical cerebrovasculardisease and one of the main diseases threatening human health and survival.Ischemia-reperfusion（I/R) injury often appears after restoration of blood supply andresults in more severe brain dysfunction, the inflammatory reaction after cerebral ischemiais also an important factor causing the secondary brain injury. Therefore,antiischemia-reperfusion injury has become the focus in the treatment of cerebral ischemia.Looking for effective drug to relieve the cerebral I/R injury is an important means toreduce the mortality and mutilation rate.Polygala japonica HOUTT (P japonica), a traditional Chinese medicinal herb hasbeen used as expectorant, ataractic，anti-inflammatory and antidepressant agents. Previousstudies showed that Polygalasaponin F (PGSF), a triterpenoid saponin monomer isolatedfrom Polygala japonica, has a very wide range of pharmacological activity in the nervoussystem, However, there were few reports about the research on its effects of anti-injury ofI/R in brain. So we chose I/R injury and associated neuroinflammation as the direction ofthis research in order to explore the protective effect of PGSF on neuronal cells injured byI/R and its mechanism.First of all, preliminary screening for pharmacological activity of PGSF was carriedout by MTT colorimetry and ELISA method in several aspects. The results show thatPGSF has markedly protective effects on PC12cells in serum-free, H2O2oxidative stress,hypoxia combined with low glucose and MPP+model of Parkinson’s disease in vitro(P<0.01, P<0.05), and can inhibit LPS induced BV-2microglia release of tumor necrosisfactor alpha(TNF-α).Then, both physical and chemical oxygen-glucose deprivation and reperfusion(OGD/R) models were established in PC12cells and primary cortical neurons respectively,and MTT assay, microscope, Hoechst33342/PI dual staining and flow cytometry methodswere used to prove that PGSF at10,1or0.1μmol/L can increase the viability of cellsinjured by OGD/R, improve the cell morphology and the nuclear morphology of PC12cells, inhibit the damage of cell membrane permeability, reduce the percentage of cell lateapoptosis or necrosis, which suggests that PGSF can protect neuronal cells against OGD/Rinjury. Furthermore，JC-1fluorescent staining, DCF-DA fluorescence probe methods and Western blot were applied to prove that the mechanism of PGSF against I/R injury may berelated to maintenance of the mitochondrial membrane potential, reducing ROS production,increasing Bcl-2/Bax protein ratio in injured cells, down-regulating expression of P53protein and active Caspase-3fragment.Finally, LPS-stimulated BV-2microglial cells neuroinflammation model was made toexplore the mechanism of PGSF antiischemia-reperfusion injury from the point of view ofthe antineuroinflammation. ELISA assay shows that PGSF can inhibit release of TNF-α,IL-1β and generation of NO in BV-2cells. Results from western blotting and reversetranscription PCR show that, PGSF can inhibit the expression of iNOS and COX-2proteinand mRNA, reduce the expression of TNF-αmRNA, IL-1β mRNA, and TLR4mRNA inBV-2cells stimulated by LPS, which suggests the antineuroinflammatory effect of PGSFmay be connected with the TLR4signal transduction pathway. In the model of PC12cells injured by LPS-stimulated BV-2microglia conditioned medium, PGSF cansignificantly increase the viability of PC12cells, which proves that PGSF can inhibit theinflammation injury to neuronal cells by inhibiting activation of microglial cells in vitro.In summary, this study demonstrates the first time that Polygalasaponin F has asignificant protective effect on neuronal cells injured by I/R, and hasantineuroinflammatory effect in vitro, which provides an important theoretical basis forrational applications and further research and development of PGSF.