Effect Of HMGB1/TLR4/NF-?B Pathway On P-glycoprotein Expression In Mouse Brain Microvascular Endothelial Cells

Multidrug resistance,one of the major causes of drug therapy failure for epilepsy,was declared to be mediated by P-glyeoprotein(P-gp)overexpression in epileptic brain tissues.However,the precise mechanism underlying the seizure-induced P-gp overexpression remains elusive.There is considerable high mobility group box 1(HMGB1)expressed and released in epileptogenic brain tissue,and it has been reported to be involved in ictogenesis.Recent study showed that HMGB1 could promote drug resistance by up-regulating P-gp expression in gastric adenocarcinoma.However,few reports are seen about the effect of HMGB1 on the overexpression of P-glycoprotein in epileptogenic tissue.Reports from different fields have verified that extracellular HMGB1 could activate its cognate receptor TLR4 and then triggers the NF-?B signaling pathway,promoting the expression of a series of inflammatory genes.Previous studys have demonstrated an exact relationship between NF-?B activation and seizure induced P-gp overexpression.Then,could HMGB1 upregulate the expression of P-gp in epileptic tissues through TLR4/NF-?B pathway? Our previous in-vivo study has demonstrated that HMGB1 could increase the expression levels of P-gp in the hippocampus of seizure rats and mice.This in-vitro study was aimed to further investigate the effects of HMGB1 on the P-gp expression in mouse brain microvascular endothelial b End.3 cells and the potential underlying mechanism.Part I Effect of HMGB1 on P-gp expression in mouse brain microvascular endothelial cellsObjective: To explore whether HMGB1 can regulate P-gp expression in mouse brain microvascular endothelial cells(BMECs)in vitro.Methods: Immortalized mouse BMEC cell line b End.3 was cultured in vitro,and was allocated into different groups.In the different time groups,b End.3 cells were treated with culture medium containing 100 ng/m L HMGB1 for 4 h,8 h,16 h,24 h and 32 h.In the different concentration groups,b End.3 cells were treated with culture medium containing 10 ng/m L,100 ng/m L and 500 ng/m L HMGB1 for 16 h.In endogenous HMGB1 over-expression group,b End.3 cells were infected with lentivirus to over-express HMGB1.In the control group,b End.3 cells were treated with culture medium without HMGB1 or infected with empty vector.The expression of P-gp-encoding gene multidrug resistance gene 1a(mdr1a)m RNA was detected by real-time quantity PCR(RT-q PCR)while P-gp and HMGB1 protein levels were detected by Western blotting and immunocytochemistry.Results: The data from RT-q PCR showed that mdr1 a mRNA expression quantity of the control group and different time groups were 1.010±0.164?2.655±0.112?2.168±0.212?1.823±0.232?1.418±0.376?1.445±0.123 while that of the control group and different concentration groups were 1.017±0.018?1.628±0.183?1.856±0.143?1.486±0.135.The expression level of mdr1 a m RNA in all time groups and concentration groups were higher than those of the control group(P<0.05).The data from Western blotting showed that,in all time groups,only 8 h and 16 h time groups had a higher P-gp protein expression than those in the control group(P<0.05),while P-gp protein expression of all concentration groups were higher than those of thecontrol group(P<0.05).The P-gp and HMGB1 protein levels of HMGB1 over-expression group were higher than those of the negative control group(P<0.05).Immunocytochemistry showed that P-gp expression was significantly higher in cells treated with HMGB1 than in control cells(P<0.01).Conclusion: HMGB1 can up-regulate mdr1 a m RNA and P-gp protein expression in brain microvascular endothelial cells,which may play a role in the drug resistance of central nervous system diseases,especially in epilepsy.Part II HMGB1 promotes P-gp expression in mouse brain microvascular endothelial cells via the TLR4/NF-?B pathwayObjective: To investigate the mechanism of HMGB1's effect on the P-gp expression in mouse brain microvascular endothelial cells.Methods: The bEnd.3 cells were divided into control group,HMGB1 treated-group,and HMGB1 treated-group intervened with toll-like receptor 4(TLR4)antagonist LPS-RS(HMGB1+LPS-RS group)or nuclear factor-kappa B(NF-?B)inhibitor SN50(HMGB1+SN50 group).In HMGB1 treated-group,b End.3 cells were treated with culture medium containing 10,100 and 500 ng/m L HMGB1.In HMGB1+LPS-RS group and HMGB1+SN50 group,cells were pre-treated with LPS-RS(1?g/ml)or SN50(50?g/ml)for 4 h before incubation with 100 ng/ml HMGB1.In the control group,cells were treated with normal medium.In immunofluorescence assay,b End.3 cells were treated with 100 ng/m L HMGB1 for 0,30,45,60 min before detection.The expression of mdr1 a m RNA and TLR4 m RNA were detected by RT-q PCR,and the P-gp,TLR4,phosphorus-NF-?B p65 and NF-?Bp65 protein levels were detected by Western blotting.NF-?B p65 subcellular distribution and activity were measured by immunofluorescence and EMSA.Results: The expression levels of TLR4 m RNA and TLR4 protein in different concentration HMGB1-treated groups were all higher than those in the control group(P<0.05).Cytoplasmic phosphorus-NF-?B p65 and nuclear NF-?B p65 level and NF-?B p65 activity were enhanced under HMGB1 treatment.As compared to control group,the expression levels of mdr1 a m RNA and P-gp,nuclear NF-?B p65 level and NF-?B p65 DNA-binding activity in HMGB1 treated-group were increased(P<0.05);As compared to HMGB1 treated-group,the above observation indexes in either HMGB1+LPS-RS group or HMGB1+SN50 group were reduced(P<0.05).Conclusions: Our data indicate that HMGB1 could enhance P-gp expression in b End.3 cells via TLR4/NF-?B pathway,suggesting that targeting the HMGB1/TLR4/NF-?B axis may be effective for inhibiting P-gp over-expression in vascular endothelial of epileptic brain tissues.