DHQ Alleviates LPS-induced Dopaminergic Neuron Damage Via Activation Of Microglial TREM2

Objective:To investigate whether dihydroquertin(DHQ)can affect lipopolysaccharide(LPS)-induced dopamine(DA)neuronal damage by regulating the activation of triggering receptor expressed on myeloid cells 2(TREM2)in microglia.Methods:(1).In vivo: male Sprague-Dawley(SD)rats(180-220 g)were randomly divided into: control,DHQ(15 mg/kg),LPS(8 μg),LPS+DHQ(5 mg/kg),and LPS+DHQ(15 mg/kg).The model of Parkinson’s disease(PD)was induced by injecting LPS into the substantia nigra of rat midbrain by stereotaxic injection,followed by continuous gavage of DHQ for one week,behavioral changes in the rats were detected by rotarod test and open field test,immunohistochemical staining was used to detect the number of tyrosine hydroxylase(TH)positive neurons,a marker of dopaminergic neurons,in the substantia nigra of the rat midbrains,and immunofluorescence staining was used to detect ionized calcium binding adaptor(IBA-1)expression,a marker of microglia,in the midbrain of rats,Western Blot was used to detect TH,IBA-1,TREM2,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-18(IL-18)protein expression expressed in the midbrain.(2).In vitro: MN9 D cells(dopaminergic neuron cell line)were randomly divided into: control,DHQ(15 μM),6-OHDA(100 μM),6-OHDA+DHQ(5 μM),and 6-OHDA+DHQ(15 μM),and 1 h after DHQ treatment,6-OHDA was added,and TH protein expression in the MN9 D cells was detected by Western Blot to observe whether DHQ directly protected DAergic neuronal damage.BV2 cells(microglial cell line)were randomly divided into: control,DHQ(15 μM),LPS(1 μg/m L),LPS+DHQ(5 μM),and LPS+DHQ(15 μM),and 1 h after DHQ treatment,LPS was added,IBA-1 and TREM2 protein expression in BV2 cells were detected by immunofluorescence double staining and Western Blot,and the contents of TNF-α,IL-1β,and IL-18 in the cell culture supernatant was detected by enzyme-linked immunosorbent assay(ELISA)kit to observe the effect of DHQ on the activation of TREM2 and neuroinflammation.To further observe whether DHQ inhibits LPS-induced neuroinflammation by increasing the activation of TREM2 in BV2 cells,TREM2 protein expression in BV2 cells was inhibited by transfection of TREM2 si RNA,and the expression of TREM2 protein was detected by Western Blot,then BV2 cells were silenced for TREM2,followed by DHQ and LPS treatment,TREM2 protein expression in the cells was detected by Western Blot,the contents of TNF-α,IL-1β,and IL-18 in the cell culture supernatant was detected by ELISA kit.Finally,BV2 cells were treated with TREM2 si RNA,DHQ,and LPS,the BV2 cell supernatant medium was transferred into MN9 D cells,and MN9 D cells were continued to be cultured,after 24 h,TH protein expression in MN9 D cells was detected by Western Blot to see if DHQ protects DAergic neurons by activating microglial TREM2.Results:(1)In vivo: Compared with the control group,the time stayed on rod and total motor distance of rats in the LPS group were significantly reduced,while DHQ(15mg/kg)significantly improved the motor dysfunction of rats.Compared with the control group,TH protein expression was significantly reduced,and IBA-1,TREM2,TNF-α,IL-1β,and IL-18 protein expression was significantly increased in the LPS group,and TH and TREM2 protein expression was increased and IBA-1,TNF-α,IL-1β,and IL-18 protein expression was significantly reduced after administration of DHQ(15 mg/kg).(2)In vitro: In MN9 D cell experiments,6-OHDA significantly reduced TH protein expression in MN9 D cells compared with the control group,and TH protein expression did not increase after DHQ(15 μM)treatment,indicating that DHQ was not able to produce a direct protective effect on DAergic neurons.In BV2 cell experiments,compared with the control group,LPS significantly increased IBA-1,TREM2,TNF-α,IL-1β,and IL-18 expression levels in BV2 cells,and after DHQ(15 μM)treatment,TREM2 protein expression increased more,and IBA-1,TNF-α,IL-1β,and IL-18 protein expression levels significantly decreased.Transfection of 40 n M TREM2 si RNA into BV2 cells,and after TREM2 silencing,DHQ(15 μM)treatment could not increase TREM2 protein expression,and could not reduce TNF-α,IL-1β,and IL-18 protein expression levels.Compared with control group,culture of MN9 D cells in the supernatant medium of LPS-treated BV2 cells significantly reduced TH protein expression,and after DHQ(15 μM)treatment,TH protein expression significantly increased,while after TREM2 silencing,DHQ(15 μM)could not increase TH protein expression,suggesting that it could not play a protective role in DAergic neurons.Conclusion: DHQ attenuates LPS-induced DAergic neuronal damage,possibly by increasing microglial TREM2 activation.