The Influence And Mechanism On The Changes Of Astrocyte And Blood-Brain Barrier Induced By LPA In Vitro

OBJECTIVE To understand if lysophosphatidic acid (LPA) couldregulate the proliferation of astrocyte (AS) and the permeability ofBlood-brain barrier(BBB).METHODS 1,The cerebral astrocyte of the neonatal SD rat werecultured in vitro and divided randomly into blank control group,small-dose dioctyl-glycerol pyrophosphate (DGPP) group, big-doseDGPP group, pertussis toxin (PTX) group, LPA group, small-dose DGPPplus LPA group, big-dose DGPP plus LPA group and PTX plus LPAgroup. The proliferation of the AS was detected by assay of MTT andFlow Cytometer (FCM); The changes of cell morphous and F-actin wereobserved by light microscope and Phalloidin; 2,To explore themechanism of AS proliferation induced by LPA futher, then the cells weredivied into blank control group, Ro31-8220 group, PMA group, LPAgroup, Ro31-8220 plus PMA group and Ro31-8220 plus LPA group. Theproliferation and morphou changes of the cells were detected by themethods mentioned above; The concentration of intra-cellular calciumion of the cells ([Ca²⁺]i) was detect by Fura-2/AM; The change of proteinkinase C-α(PKC-α) and hypoxia inducible factor-1α(HIF-1α) inside ASwere detected by Western blot; 3,The BBB models were dividedrandomly into blank control group, LPA group and Ro31-8220 plus LPAgroup. The permeability of BBB was detected byγ-events-per-unit-timemete; The tight-junction was observed by electronmicroscope. The cellsexpressing PKC-αwere detected by FCM. The claudin-5 was detected byWestern blot.RESULTS 1,LPA increased the survival rate and the S-stage proportion and stimulate morphous change of AS as compared with blankcontrol group (P＜0.01), and 10μmol/L LPA was the most effectconcentration; 2,DGPP and PTX could block the effection of LPA thatmentioned above in comparation with LPA group (P＜0.01) and thebig-dose DGPP was more effective than small-dose DGPP (P＜0.01); 3,LPA increased the concentration of [Ca²⁺]_i and the expression of PKC-αand HIF-1αas compared with blank control group (P＜0.01). The indexesof the concentration of [Ca²⁺]_i and PKC-αin Ro31-8220+LPA groupwere decresed except the expression of HIF-1αas compared with the LPAgroup(P＜0.01); 4,LPA increased the permeability of ¹²⁵I-BSA of BBBand the expression of PKC-αbut claudin-5 as compared with the blankcontrol group (P＜0.01). LPA also open Tight junction (TJ) and regulatethe recombination of F-actin simultaneously. But pretreated by Ro31-8220, the effection of LPA mentioned above were attenuated ascompared with the LPA group (P＜0.01).CONCLUSIONS 1,LPA could accommodate the proliferationand the morphous change of AS through LPA_1/3-G_i/o protein coupledreceptors which may related with the activation of PKC-α; 2,LPA couldbreak the conjunction of TJ and increase the permeability of BBB via theactivation of PKC-αsignal channel which down regulation of claudin-5and recombination of F-actin in BMEC.