Abstract：OBJECTIVE: To study the influence of lipopolysaccharide (LPS) on the permeability of rat brain microvascular endothelial cells (BMECs) and possible molecular mechanism. METHODS: Monolayers of primary rat BMECs were separated and cultured, and then treated with (LPS group) or without LPS (control group). The barrier integrity was measured by transendothelial electrical resistance (TEER) assay. The degrees of RhoA activation were determined by Pull-down assay. The expression levels of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins were detected by Western blot analysis. RESULTS: The average TEER values of rat BMECs in the LPS group were 108.3±4.2 Ω?cm2 and 85.4±2.5 Ω?cm2 respectively 3 and 12 hrs after LPS treatment, which were significantly lower than that in the control group (159.0±8.6 Ω?cm2). Compared with the control group, the activity of RhoA started to increase 5 minutes after LPS treatment, and the expression of p115RhoGEF protein started to increase 1 hr after LPS treatment and the cellular protein levels of ZO-1, occludin and claudin-5 decreased significantly 3 hrs after LPS treatment in the LPS group (P<0.05). CONCLUSIONS: LPS may activate the p115RhoGEF/RhoA pathway and decrease protein expression of ZO-1, occludin and claudin-5, resulting in an increased permeability of rat BMECs.
DENG Xiao-Lu,HE Fang,PENG Jing et al. Influence of lipopolysaccharide on the permeability of rat brain microvascular endothelial cells and the molecular mechanism[J]. CJCP, 2011, 13(11): 908-911.
［3］Holinstat M, Mehta D, Kozasa T, Minshall RD, Malik AB. Protein kinase C alpha-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement［J］. J Biol Chem, 2003, 278(31):28793-28798.
［5］Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, et al. Occludin: a novel integral membrane protein localizing at tight junctions［J］.J Cell Biol, 1993, 123(6 Pt 2):1777-1788.
［6］Ohtsuki S, Sato S, Yamaguchi H, Kamoi M, Asashima T, Terasaki T. Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells［J］. J Cell Physiol, 2007, 210(1):81-86.
［7］Chen F, Ohashi N, Li W, Eckman C, Nguyen JH. Disruptions of occludin and claudin-5 in brain endothelial cells in vitro and in brains of mice with acute liver failure［J］. Hepatology, 2009, 50(6):1914-1923.
［8］Sheth P, Delos Santos N, Seth A, LaRusso NF, Rao RK. Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism［J］. Am J Physiol Gastrointest Liver Physiol, 2007, 293(1):G308-G318.
［9］Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase)［J］. J Biol Chem, 1996, 271(34):20246-20249.
［9］Hart MJ, Sharma S, elMasry N, Qiu RG, McCabe P, Polakis P, et al. Identification of a novel guanine nucleotide exchange factor for the Rho GTPase［J］. J Biol Chem, 1996, 271(41): 25452-25458.
［10］Birukova AA, Birukov KG, Smurova K, Adyshev D, Kaibuchi K, Alieva I, et al. Novel role of microtubules in thrombin-induced endothelial barrier dysfunction［J］. FASEB J, 2004, 18(15):1578-1590.
［11］Wettschureck N, Offermanns S. Rho/Rho-kinase mediated signaling in physiology and pathophysiology［J］. J Mol Med, 2002, 80(10):629-638.
［12］Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, et al. Regulation of tight junction permeability and occludin phosphorylation by RhoA-p160ROCK-dependent and -independent mechanisms［J］. J Biol Chem, 2001, 276(13):10423-10431.
