Huaming Xu1, Yuan Liu2, Dongsheng Wang3, Zhenqiang Zhang4. 1. Department of Integrated Traditional and Western Medicine, Xiangya Hospital of Central South University, No.87 Xiangya Road, Changsha, Hunan Province, 410008, China; Henan University of Chinese Medicine, No.156 Jinshui East Road, Zhengzhou, Henan Province, 450046, China. Electronic address: sirxu@foxmail.com. 2. Beijing Hongci Healthcare Investment Management Co., Ltd., 89 Jinbao Street, Dongcheng District, Beijing, 100005, China. Electronic address: hellokitty_2000@163.com. 3. Department of Integrated Traditional and Western Medicine, Xiangya Hospital of Central South University, No.87 Xiangya Road, Changsha, Hunan Province, 410008, China. Electronic address: wdsh666@126.com. 4. Henan University of Chinese Medicine, No.156 Jinshui East Road, Zhengzhou, Henan Province, 450046, China. Electronic address: zhang_zhenqiang@126.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Shenmai injection (SMI), a traditional Chinese herbal medicine is widely used for the clinical treatment of cerebral infarction in China. AIM OF THE STUDY: Tight junctions (TJs) are major components of the blood-brain barrier (BBB) that physically restrict the paracellular diffusion of blood-borne substances between endothelial cells into the CNS. TJ proteins are associated with cholesterol-enriched regions of plasma membrane known as lipid rafts, which are critical for the trafficking, positioning and function of TJ proteins. In this study, we investigated the effect of SMI on the expression and trafficking of the key TJ-associated protein, occludin, in lipid rafts. MATERIALS AND METHODS: Using a neutral pH, rat cerebral microvessels were subjected to detergent-free density-gradient fractionation to isolate lipid rafts containing occludin. Transmission electron microscopy (TEM) was performed to study the effects of drug administration on ultrastructural changes to TJs. Western blotting (WB), immunofluorescence (IF), and co-immunoprecipitation (COIP) were used to observe the localization and function of TJ-associated proteins. RESULTS: We successfully isolated cerebral microvessels and separated lipid rafts from plasma membranes. With SMI treatment, extravasation of FITC-albumin decreased around the cerebral vessels by IF, the tight junctions were found to still be intact and the basement membrane appeared to be of uniform thickness in TEM. Compared with the untreated group, the co-expression of flotillin-1 and occludin in microvascular endothelial cells was increased and distributed continuously in SMI treatment as shown in double label IF. SMI significantly increased the translocation of occludin to lipid raft fractions by WB and COIP. CONCLUSIONS: SMI helps maintain the proper assembly of the TJ multiprotein complex in lipid rafts, thereby helping to preserve BBB functional integrity during focal cerebral ischemic insult. Our findings enhance our understanding of the mechanisms underlying the neuroprotective effect of SMI in cerebral ischemia.
ETHNOPHARMACOLOGICAL RELEVANCE: Shenmai injection (SMI), a traditional Chinese herbal medicine is widely used for the clinical treatment of cerebral infarction in China. AIM OF THE STUDY: Tight junctions (TJs) are major components of the blood-brain barrier (BBB) that physically restrict the paracellular diffusion of blood-borne substances between endothelial cells into the CNS. TJ proteins are associated with cholesterol-enriched regions of plasma membrane known as lipid rafts, which are critical for the trafficking, positioning and function of TJ proteins. In this study, we investigated the effect of SMI on the expression and trafficking of the key TJ-associated protein, occludin, in lipid rafts. MATERIALS AND METHODS: Using a neutral pH, rat cerebral microvessels were subjected to detergent-free density-gradient fractionation to isolate lipid rafts containing occludin. Transmission electron microscopy (TEM) was performed to study the effects of drug administration on ultrastructural changes to TJs. Western blotting (WB), immunofluorescence (IF), and co-immunoprecipitation (COIP) were used to observe the localization and function of TJ-associated proteins. RESULTS: We successfully isolated cerebral microvessels and separated lipid rafts from plasma membranes. With SMI treatment, extravasation of FITC-albumin decreased around the cerebral vessels by IF, the tight junctions were found to still be intact and the basement membrane appeared to be of uniform thickness in TEM. Compared with the untreated group, the co-expression of flotillin-1 and occludin in microvascular endothelial cells was increased and distributed continuously in SMI treatment as shown in double label IF. SMI significantly increased the translocation of occludin to lipid raft fractions by WB and COIP. CONCLUSIONS:SMI helps maintain the proper assembly of the TJ multiprotein complex in lipid rafts, thereby helping to preserve BBB functional integrity during focal cerebral ischemic insult. Our findings enhance our understanding of the mechanisms underlying the neuroprotective effect of SMI in cerebral ischemia.