Transport Of Plumbagin And Lapachol On In Vitro BBB Model And Effect On Endothelial And Glioma Cells

Naphthoquinone from natural source is an important class of active ingredients andwidespread in nature. The plumbagin and lapachol are naphthoquinone compoundsand belong to alpha-(1,4-) type of naphthoquinone. And the structure determinestheir unique physical and chemical properties and pharmacological effects. Theplumbagin has numerous biological activities, including anti-tumor, anti-oxidationand so on. And lapachol has been widely used as an anti-malarial drug. In this paper,we systematically expounded that the toxicities and protective effect of plumbaginand lapachol to rat brain microvascular endothelial cells (rat BMEC), the transport inthe blood-brain barrier in vitro, and the proliferation inhibition of drugs to gliblastomaC6cells. According to these results, we could provide pharmacological evidences forbetter utilized of plumbagin and lapachol in clinical studies.1. Transport of plumbagin and lapachol on BBB in vitroRat BMEC and AS were primary cultured and passaged. The fourth generation ofrat BMEC and AS were co-cultured on24-well cell culture inserts to build BBBmodel in vitro. After cultured for10d the tight junction of rat BMEC was formed. Thecytotoxicity of plumbagin and lapachol on rat BMEC and the safe concentration oftransporter inhibitors were determined by MTT assay. Ultimately the appropriateconcentrations were chosen based on the solubility and nontoxic concentrations ofdrugs. Then the influences of transporter inhibitors on transport of plumbagin andlapachol were studied. The samples were tested by HPLC and Pappwas calculated toobtain the efflux ratio. The results turned out that probenecid and novobiocin could restrain the active efflux of MRP2and BCRP transporters to increase the permeabilityof plumbagin and lapachol.2. The protective effect of plumbagin and lapachol to rat BMECThe fourth generation of rat BMEC was used to build the hypoxia injury model ofrat BMEC to simulate local acute cerebral ischemia in human body by oxygen andglucose deprivation (OGD). To determine appropriate OGD timepoint, the viability ofrat BMEC was detected via MTT assay after disposed by OGD. Ultimately theappropriate concentrations were chosen based on the solubility and nontoxicconcentrations of drugs and the lactate dehydrogenase (LDH) leakage tests wereproceeded. The fourth generation of rat BMEC was inoculated on24-well cultureplates. And normoxia control group, OGD control group and medicationadministration groups of different concentrations were set and incubated respectivelyin ordinary incubator and low oxygen incubator. Then the supernatant and cell lysisbuffer were tested by LDH quantification kits and the LDH leakage rates werecalculated. The results showed that the LDH leakage rate of OGD group increasedsignificantly compared with normoxia control group. The plumbagin group showed acertain degree of cytotoxicity when the concentration was10μM and the LDHleakage rate was higher compared with normoxia control group. The LDH leakagerate when the concentration of plumbagin was5μM was not significantly differentcompared with OGD control group. But plumbagin could reduce the cellular damageafter OGD when the concentration was1μM. In addition, the LDH leakage rate oflapachol when the concentration was100μM was not significantly different comparedwith OGD control group. But lapachol could alleviate the cellular injury induced byOGD when the concentrations were10μM and1μM.3. Proliferation inhibition of plumbagin and lapachol to glioblastoma C6cellThe twentieth generation of glioblastoma C6cells was inoculated on96-well culture plates. After incubated for24hours the cells were exposed to plumbagin andlapachol. The culture plates were incubated for another48hours and then theviabilities of glioblastoma C6cells were detected via MTT assay to study theproliferation inhibition of drugs to C6cells. In the mean time, a listed anti-tumormedicine temozolomide was chosen as positive control. The results indicated that theproliferation inhibition to C6cells of plumbagin when the concentration was100μMequaled the proliferation inhibition of temozolomide when the concentration was500μM, while the proliferation inhibition of lapachol was weaker compared withplumbagin and temozolomide. In addition, the proliferation inhibition on C6cells stillexisted when the medium and low concentrations of lapachol were utilized to C6cells.