Lalita Subedi1, Bhakta Prasad Gaire2, Moon Ho Do1, Taek Hwan Lee3, Sun Yeou Kim4. 1. Laboratoy of Pharmacognosy, College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, 406-799, Republic of Korea. 2. Laboratory of Neuropharmacology, College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, 406-799, Republic of Korea. 3. College of Pharmacy, Yonsei University, #162-1 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea. 4. Laboratoy of Pharmacognosy, College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, 406-799, Republic of Korea. Electronic address: sunnykim@gachon.ac.kr.
Abstract
BACKGROUND: Lindera neesiana Kurz (Lauraceae), popularly known as Siltimur in Nepal, is an aromatic and spicy plant with edible fruits. It is a traditional herbal medicine widely used for the treatment of diarrhea, tooth pain, headache, and gastric disorders and is also used as a stimulant. PURPOSE: The aim of the present study was to examine in vitro cytoprotective, anti-neuroinflammatory and neuroprotective potential of an aqueous extract of L. neesiana (LNE) fruit using different central nervous system (CNS) cell lines. METHODS: In order to study the neuroprotective potential of LNE, we used three different types of CNS cell lines: murine microglia (BV2), rat glioma (C6), and mouse neuroblastoma (N2a). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, and prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and nerve growth factor (NGF) release in the culture media was determined using enzyme linked immunosorbent assay (ELISA) kits. Western blot analysis was performed to determine the protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), mitogen activated protein kinase (MAPK) family proteins, Bax, B cell lymphoma (BCL)-2, and cleaved caspase 3. Neurite outgrowth was determined using the IncuCyte imaging system. RESULTS: LNE treatment not only reduced nitric oxide (NO) production in a dose-dependent manner, but also significantly reduced proinflammatory cytokines, iNOS and COX-2 production by lipopolysaccharide (LPS) stimulated BV-2 cells. LNE increased the expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), whereas p-p38 and p- janus kinase (JNK) expression was significantly decreased in activated microglia. Furthermore, LNE increased cell viability of N2a cells, which was accompanied by decreased caspase-3 expression and the ratio of Bax/Bcl2 protein expression as well as increased NGF and neurite outgrowth, suggesting its neuroprotective potential against LPS-induced effects. Additionally, LNE substantially increased nuclear factor erythroid 2-related factor 2 (Nrf2) secretion in N2a cells and inhibited lipid dehydrogenase (LDH) release in H2O2-stimulated BV2 cells demonstrating the strong anti-inflammatory and antioxidant effects of LNE in CNS cell lines. CONCLUSION: Here we found that water the soluble extract of LNE has promising anti-neuroinflammation and anti-apoptotic properties and identify LNE as a potential natural candidate for neuroprotection.
BACKGROUND: Lindera neesiana Kurz (Lauraceae), popularly known as Siltimur in Nepal, is an aromatic and spicy plant with edible fruits. It is a traditional herbal medicine widely used for the treatment of diarrhea, tooth pain, headache, and gastric disorders and is also used as a stimulant. PURPOSE: The aim of the present study was to examine in vitro cytoprotective, anti-neuroinflammatory and neuroprotective potential of an aqueous extract of L. neesiana (LNE) fruit using different central nervous system (CNS) cell lines. METHODS: In order to study the neuroprotective potential of LNE, we used three different types of CNS cell lines: murine microglia (BV2), ratglioma (C6), and mouseneuroblastoma (N2a). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, and prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and nerve growth factor (NGF) release in the culture media was determined using enzyme linked immunosorbent assay (ELISA) kits. Western blot analysis was performed to determine the protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), mitogen activated protein kinase (MAPK) family proteins, Bax, B cell lymphoma (BCL)-2, and cleaved caspase 3. Neurite outgrowth was determined using the IncuCyte imaging system. RESULTS:LNE treatment not only reduced nitric oxide (NO) production in a dose-dependent manner, but also significantly reduced proinflammatory cytokines, iNOS and COX-2 production by lipopolysaccharide (LPS) stimulated BV-2 cells. LNE increased the expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), whereas p-p38 and p- janus kinase (JNK) expression was significantly decreased in activated microglia. Furthermore, LNE increased cell viability of N2a cells, which was accompanied by decreased caspase-3 expression and the ratio of Bax/Bcl2 protein expression as well as increased NGF and neurite outgrowth, suggesting its neuroprotective potential against LPS-induced effects. Additionally, LNE substantially increased nuclear factor erythroid 2-related factor 2 (Nrf2) secretion in N2a cells and inhibited lipid dehydrogenase (LDH) release in H2O2-stimulated BV2 cells demonstrating the strong anti-inflammatory and antioxidant effects of LNE in CNS cell lines. CONCLUSION: Here we found that water the soluble extract of LNE has promising anti-neuroinflammation and anti-apoptotic properties and identify LNE as a potential natural candidate for neuroprotection.