The Effect Of Ginsenosides Rb1 And Rg1 On Mitochondria Of Astrocytes Damaged By OGD/R And Its Mechanism

Cerebral ischemia-reperfusion injury(CIRI)is one of the leading causes of death and disability for millions of people in the world.When ischemia occurs locally in the brain,blood supply to the brain will be partly interrupted as a result of thrombosis,embolism,and causing ischemic damage.When blood is perfused,it will be accompanied by a large amount of oxygen to generate reactive oxygen species(ROS),and causing secondary damage.The pathological mechanism of CIRI is complicated and involves a series of pathological cascade reactions,for example,oxidative stress will damage mitochondrial DNA(mtDNA),cause mitochondrial dysfunction,release cytochrome C,activate various signal pathways,and causes apoptosis.Astrocytes(AS)are the most extensive type of glial cells among nerve cells,it is essential for the processes required for the formation and maintenance of the blood-brain barrier,neural circuit function,synapse formation,nerve transmission,metabolic regulation,and normal biological function of the central nervous system.During cerebral ischemia,neurons are damaged first.When the energy from oxidative phosphorylation(OXPHOS)and glycolysis is insufficient,adenosine triphosphate(ATP)produced by astrocytes is uptaked by neurons,thereby protecting neuronal cells from ischemic damage.Ginsenosides Rb1(20-S-protopanaxadiol aglycon,Rbl)and Rg1(20-S-protopanaxatriol aglycon,Rg1)are the main active ingredients.Recent studies have shown that ginsenosides Rbl and Rg1 have various pharmacological effects such as improving microcirculation,antithrombotic,anti-inflammatory,scavenging oxygen species and antioxidation.Ginsenosides Rbl and Rg1 have certain therapeutic effects on immune diseases,cardiovascular and cerebrovascular diseases,cancer,tumors,etc.They also have certain protective effects on CIRI.This study established oxygen-glucose deprivation/reoxygenation(OGD/R)model to mimic the cerebral ischemia-reperfusion pathological process in vitro and evaluate whether ginsenosides Rb1 and Rg1 have mitochondrial protective effects against OGD/R-induced injury in primary mouse astrocytes.Aims:This study aimed to evaluate the neuroprotective effects of ginsenosides Rbl and Rg1and to explore the mechanisms involved.Methods:ICR mice(within 24 h after birth)were taken for primary astrocyte culture,the third generation of near-fused cells were used for the experiments.Astrocytes were treated with normal conditions,OGD/R,OGD/R plus Rbl,or OGD/R plus Rg1.1.Effects of ginsenosides Rbl and Rgl on OGD/R-treated astrocytes(1)GFAP immunofluorescence staining validated primary cultured mouse astrocytes.(2)Cell viability was measured to evaluate the cytotoxicity of Rbl and Rgl by CCK-8 assay.(3)Intracellular ROS and catalase(CAT)were detected to evaluate oxidative stress by ROS assay kit and CAT analysis kit.2.Protective mechanism of ginsenosides Rbl and Rgl on OGD/R-injured astrocyte mitochondria.(1)The mitochondrial DNA copy number and mitochondrial membrane potential(MMP)were measured to evaluate mitochondrial function by real-time quantitative polymerase chain reaction(PCR)and JC-1 dye kit.(2)The level of cellular ATP and the activities of the mitochondrial respiratory chain complexes ?-? were measured to evaluate OXPHOS levels by mitochondrial respiratory chain complexes kits and ATP detection assay Kit.Results:1.Ginsenosides Rbl and Rgl attenuated OGD/R-induced injury in astrocytes.(1)GFAP immunofluorescence staining showed that cells in culture appear to express high levels of GFAP and the purity of the cultures was high.(2)Compared with control cells,the cell viability was significantly decreased in OGD/R-treated astrocytes(P<0.01).After 6 h of OGD,astrocytes were reoxygenated for 24 h,Rbl and Rgl at 5 and 10 ?M concentrations significantly increased the cell viability(P<0.01).2.Rbl and Rg1 suppressed ROS production and increased CAT activity in OGD/R-treated astrocytesCompared with control cells,the production of intracellular ROS was significantly enhanced in OGD/R-treated astrocytes(P<0.01),but CAT activity was decreased significantly(P<0.01),ginsenosides Rbl(5 ?M)and Rg1(10?M)administration significantly reduced the production of ROS(P<0.01)and increased CAT activity(P<0.05)compared to OGD/R-treated astrocytes.3.Ginsenosides Rbl and Rg1 improve mitochondrial function of astrocytes by regulating mitochondrial oxidative phosphorylation(1)Compared to control cells,after OGD/R,MMP depolarization was continuously enhanced(P<0.01)and mtDNA content was decreased(P<0.01)in astrocytes under OGD/R conditions.Rbl(5 ?M)and Rg1(10 ?M)administration significantly attenuated the MMP depolarization(P<0.01)and increased mtDNA copy number(P<0.05),compared to that in OGD/R-treated astrocytes.(2)Compared with control cells,after OGD/R,the activities of complexes ?,?,?,? and the level of ATP were significantly decreased in OGD/R-treated astrocytes(P<0.05).Incubation with Rbl(5 ?M)and Rg1(10 ?M)increased the activities of complexes ?,?,?,and ?(P<0.05)and ATP levels(P<0.05),but there was no significant difference in complex ? activity among the experimental groups.Conclusion:In conclusion,our findings indicate that ginsenosides Rbl and Rg1 are endowed with a significant protective mitochondrial function against OGD/R-induced damage in astrocytes.The mechanism may be related to improve efficiency of mitochondrial oxidative phosphorylation and reduce oxidative stress.