The Effect Of Panax Notoginseng Saponins On Neuroinflammation In Aging Rats Through TXNIP Mediated NLRP3 Inflammasome Activation

Objective To study the effect of Panax notoginseng saponins(PNS)on neuroinflammation in aging rats through thioredoxin interacting protein(TXNIP)mediated NLRP3 inflammasome activation.To provide theoretical basis and laboratory basis for the prevention and treatment of aging-related neurodegenerative diseases.Methods In vivo animal experiments: 18-month-old male SD rats(SPF grade)were randomly divided into aging group(24M),PNS low-dose group(10 mg/kg)and PNS high-dose group(30 mg/kg),with 15 rats in each group.The PNS groups were given 10mg/kg and 30 mg/kg daily for six months.From the second month of administration,another 15 2-month-old rats were purchased as youth control,named as 6-month-old group(6M),6M and 24 M group rats fed normally.(1)The effect of PNS on the cellular morphological structure of cerebral cortex and hippocampal CA1 and CA3 regions of rats was observed using HE staining.The effect of PNS on the number of microglia in cortex and hippocampal CA1 and CA3 regions of rats were detected by immunofluorescence.The co-localization of NOD-like receptor 3(NLRP3)and TXNIP with microglial marker Iba-1in the cortex and hippocampal CA1 and CA3 regions of rats were detected by immunofluorescence.The effect of PNS on the expression of neuroinflammatory factor interleukin-1?(IL-1?),inflammasome associated protein NLRP3,cysteinyl aspartate specific proteinase-1(Caspase-1),apoptosis-associated speck-like protein containing CARD(ASC)and TXNIP protein in cortex and hippocampus of aging rats were detected by Western Blot.In vitro cell experiments:(1)The model of NLRP3 inflammasome activation was established by co-stimulation of lipopolysaccharide(LPS)and adenosine triphosphate(ATP)to observe the protective effect of PNS.The cells were divided into normal control group(Control),model group(LPS+ATP),PNS low dose group(25 ?g/mL)and PNS high dose group(50 ?g/mL).The concentration of LPS was 1 ?g/mL for 6 hours and ATP was 2mmol/L for 30 minutes.The effects of PNS on the viability of BV2 microglia was detected by MTT.The effects of PNS on the expression of IL-1?,NLRP3,ASC,Caspase-1 and TXNIP protein were detected by Western Blot and immunofluorescence.(2)After adding verapamil,an inhibitor of TXNIP,BV2 microglia were co-stimulated with LPS and ATP.The expression levels of IL-1?,NLRP3,ASC and Caspase-1 protein was detected by Western Blot and immunofluorescence.The effect of TXNIP and PNS on the content ofreactive oxygen species(ROS)in BV2 microglia induced by LPS and ATP co-stimulation was detected by ROS fluorescence probe-DHE method.Results In vivo animal experiments:(1)Compared with 6M group,the neurons in the cortex and hippocampal CA1 and CA3 regions of 24 M group were disordered,the cell volume became smaller and the cell staining was deeper,and the cell body was irregular.Compared with 24 M group,the neurons in PNS group(10 mg/kg and 30 mg/kg)were arranged neatly,and the cells were lightly stained and the cell bodies were round.Immunofluorescence results showed that the number of microglia in the cortex and hippocampal CA1 and CA3 regions of 24 M group was significantly more than that in 6M group,while the number of microglia decreased after the intervention with PNS(10 mg/kg and 30 mg/kg).Meanwhile,NLRP3 and TXNIP were co-located with microglia in the cortex,hippocampal CA1 and CA3 regions of rats.Western Blot results showed that the protein levels of IL-1?,NLRP3,ASC,Caspase-1 and TXNIP protein in cortex and hippocampus of rats in 24 M group were significantly higher than that in 6M group(P<0.05 or P<0.01).After intervention with PNS(10 mg/kg and 30 mg/kg),the protein levels of the above proteins were down-regulated in each dose group(P<0.05 or P<0.01).In vitro cell experiments:(1)MTT results showed that PNS(6.25 ?g/mL,12.5 ?g/mL,25 ?g/mL and 50 ?g/mL)had no effect on the viability of BV2 microglia in comparison with the normal control group.However,the cell viability was decreased significantly at the concentration of 200 ?g/mL(P<0.01).Therefore,we chose the concentration of 6.25?g/mL,12.5 ?g/mL,25 ?g/mL and 50 ?g/mL to detect the effect of PNS on the activity of BV2 microglia induced by LPS and ATP co-stimulation.Compared with the normal control group,the viability of BV2 microglia in LPS+ATP stimulated group was significantly decreased(P<0.01).After intervention with different concentrations of PNS(6.25 g/mL,12.5 g/mL,25 g/mL and 50 g/mL),the viability of BV2 microglia was increased at all concentrations of PNS(P<0.05 or P<0.01).Western Blot and immunofluorescence results showed that PNS(25 ?g/ml and 50 ?g/mL)could decrease the expression of IL-1?,NLRP3,ASC,Caspase-1 and TXNIP protein in LPS+ATP stimulated group(P<0.05 or P<0.01).(2)After,microglia were stimulated by LPS and ATP with Verapamil,an inhibitor of TXNIP.Western Blot and immunofluorescence results showed that the protein levels of IL-1?,NLRP3,ASC and Caspase-1 decreased in the model group after TXNIP was inhibited(P<0.05,P<0.05 or P<0.01).Compared with the normal control group,the ROS in BV2 microglia increased significantly after LPS and ATP co-stimulation while the ROSin BV2 microglia decreased significantly after TXNIP was inhibited.Conclusion Panax notoginseng saponins can attenuate the neuroinflammatory response in the cortex and hippocampus of aging rats.Panax notoginseng saponins can reduce the expression levels of NLRP3 inflammasome and inflammatory factors,decrease the production of reactive oxygen species,and have a good anti-inflammatory effect.The mechanism may be related to activation of NLRP3 inflammasome mediated by TXNIP signaling pathway.