Ginsenoside Rb1 Attenuates Lipopolysaccharide-induced Brain Injury In Mice And Its Mechanism Of Action

In the mammalian central nervous system(CNS),astrocytes and microglia play important roles in maintaining the functional stability of the CNS.Under pathological conditions,abnormal astrocyte function occurred.On the one hand,glutamate(Glu)released by neural cells into the synaptic cleft cannot be taken up into astrocytes with high efficiency,resulting in abnormally higher glutamate concentration in the synaptic cleft.On the other hand,the impaired ability of astrocytes to convert Glu for glutamine(Gln)synthesis,destroying the Glu-Gln cycle that is highly dependent on astrocytes,the impairment of the functions of astrocytes,such as the regulation of nervous transmitter,the construction of blood brain barrier and the release of neuronutrition factors.This would cause astrocytes to shift from an anti-inflammatory state to a pro-inflammatory state,interacting with activated microglia and causing further damage to neural cells.Therefore,maintenance of the Glu-Gln recycling function,protection of astrocytes,and inhibition of microglial activation are all important strategies to attenuate neural injury.Ginseng,as a traditional Chinese medicinal material,has accumulated a large number of studies.ginsenoside Rb1,one of its important ginsenosides,can alleviate ischemic brain injury,reduce neuroinflammation,prevent seizures and so on.Our group previously showed that,in vitro,ginsenoside Rbl promotes the mRNA and protein expression of glutamate transporter-1(GLT1),glutamine synthase(GS),and exerts protective effects on astrocytes.However,is ginsenoside Rb1 still effective in regulating GLT1/GS under CNS inflammatory conditions?Is it associated with the reduction of neuroinflammation and the protection of nerve cells?These are the main aims of this study.Aims:Surrounding astrocytes and microglia,this study aimed to evaluate the neuroprotective effects of ginsenosides Rb1 under neuroinflammatory conditions and to explore the mechanisms involved.Methods:Six-week-old male ICR mice were randomly assigned into six groups:the control group,the LPS group,the ginsenoside low-dose group(20 mg/kg/d,Rb1 20+LPS),ginsenoside high dose group(40 mg/kg/d,Rb1 40+LPS),dexamethasone(3 mg/kg/d,Dex+LPS)and edaravone group(10 mg/kg/d,EDA+LPS).To create a systemic inflammatory model,a single intraperitoneal injection of 3 mg/kg LPS(lipopolysaccharide,LPS)dissolved in normal saline was given to each mouse within 15-30 min after the last injection of the drug.The control group mice were injected with an equal volume of normal saline.1 Effects of ginsenoside Rbl on behavioral impairment and neuronal damage in LPS mice(1)The open field test and the beam walking test were measured to evaluate the effects of ginsenoside Rbl on behavior changes within 20?24 h after LPS injection.(2)The somatosensory cortex and hippocampus regions of LPS mice brains by NeuN immunofluorescence staining were measured to the neuroprotective effect of ginsenoside Rb1.2 Effects of ginsenoside Rbl on microglia and astrocytes in the LPS mice(1)IBa-1 immunofluorescence staining and ELISA were measured to the effects of ginsenoside Rb1 on microglia activation and production of inflammatory factor IL-1? in LPS mice.(2)Western blots and double immunofluorescence staining for GLT-1 and GS/GFAP were measured to the effect of ginsenoside Rbl on glutamate regulation.Results:1 Rb1 improves behavior deficits induced by LPS.(1)In the open-field test.Compared to control mice,a reduction of voluntary locomotor activity as indicated by slow walking and decreased activity was observed in mice treated with LPS within 24 hours.Rb1 treatment improved the abnormal behavior caused by LPS,which was manifested as prolonged movement distance and increased movement speed.The results conclusively indicate that Rb1 markedly relieves the behavior changes caused by LPS and improves locomotive activity.(2)In the beam walking test.There were no foot slips or falls observed in control mice.However,LPS mice were becoming slow when crossing the beam,and a significant number of foot slips or falls were observed in LPS-treated mice.Some of the LPS treated mice were unable to cross the beam and some could not even place their limbs on the beam.Compared to LPS mice,the mice in the 20 mg/kg and 40 mg/kg groups had a much better performance crossing the beam with fewer foot slips.There were no falls observed in both the Rb1 20 mg/kg and 40 mg/kg groups.The results suggest that Rb1 improves the motor coordination of LPS treated mice.2 Rbl attenuates LPS-induced neuron injury in the brain.LPS treatment mildly reduced the NeuN in S1Tr,S1BF and S2 areas of the somatosensory cortex and CA1 in the hippocampus compared to the control mice.Rb1 20 mg/kg and 40 mg/kg treatment significantly attenuated LPS induced neuronal loss.However,Rb1 40 mg/kg did not show a greater effect than 20 mg/kg.The above immunostaining results indicate that Rb1 protects neurons from LPS-induced damage.3 Rb1 suppresses LPS-induced microglial activation and IL-1? production in the brain.LPS induced an obvious microglial activation with thicker protrusions and larger cell soma.The IL-1? in the brain of LPS mice was significantly higher than that in control mice.Rb1 treatment significantly suppressed the microglial activation in CA1,S1Tr,S1BF and S2 areas,and inhibited the IL-1? production induced by LPS.The results suggest that Rbl effectively attenuated the LPS-induced microglial activation and IL-1?production.4 Rbl enhances the expression of GLT-1 and GS in the brain of LPS mice.systemically LPS administration led to reactive astrogliosis in hippocampus CA1 that was characterized by hypertrophy and an increasing number of GFAP-labelled cells along with considerable expression of GLT-1 transporter.However,the expression of GS was reduced after LPS treatment.Rb1 20 mg/kg and 40 mg/kg treatment further enhanced the production GLT in astrocytes after LPS insult,and significantly attenuated the GS reduction induced by LPS,which was confirmed by both immunostaining and Western Blot analysis.The results reveal that Rb1 can further increase the expression of GLT-1 and attenuate the GS reduction induced by LPS insult.Conclusion:Ginsenoside Rb1 improved LPS induced behavioral abnormalities in mice and protect neuronal cells in sensorimotor areas of the cerebral cortex and hippocampus in LPS mice.Besides,Rb1 regulated GLT-1 and GS expression under neuroinflammatory conditions,and inhibited microglial activation.It is presumed that the neuroprotective effect of ginsenoside Rb1 in LPS mice is closely related to the regulation of astrocyte GLT-1/GS expression,which then improves the Glu/Gln cycle.