Triptolide inhibits TNF-a, IL-1b and NO production in primary microglial cultures Hui-Fang Zhou, Dong-Bin Niu, Bing Xue, Feng-Qiao Li, Xian-Yu Liu, Qi-Hua He, Xin-Hong Wang and Xiao-Min Wang CA Neuroscience Research Institute, Peking University, 38 Xueyuan Road, Beijing 100083, PR China CA Corresponding Author: xmwang@bjmu.edu.ch Received 4 December 2002; accepted18 February 2003 DOI: 10.1097/01.wnr.0000073682.00308.47 Microglia are believed to participate in the mediation of neuro- degeneration through producing a variety of cytotoxic factors upon activation. Pharmacological intervention in microglial activa- tion may therefore exert a neuroprotective e¡ect. In exploring pharmacological agents that can a¡ect microglial activation, we found in this study that triptolide possesses a powerful inhibitory in£uence over microglia. Pretreatment with triptolide was able to dose-dependently reduce the lipopolysaccharide (LPS)-induced nitrite accumulation and tumor necrosis factor-a and interleukin- 1b release from LPS-activated microglia as revealed by Griess reac- tion and ELISA, respectively. Triptolide reduced LPS-stimulated mRNA expression of all three in£ammatory factors. The results obtained from this study demonstrate that triptolide can inhibit in£ammatory responses of microglia to in£ammatory stimulation via a mechanism involving the inhibition of the synthesis and re- lease of in£ammatory factors. NeuroReport 14 :1091^1095  c 2003 Lippincott Williams & Wilkins. Key words: In£ammation; Microglia; Neuroprotection; Nitric oxide; Proin£ammatory cytokines; Triptolide; Tripterygium wilfordii Hook F. INTRODUCTION Inflammation in the central nervous system is closely associated with the pathogenesis of various neurodegener- ative diseases, including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis and AIDS dementia complex [1]. The hallmark of brain inflammation is the activation of microglia [2]. Upon activation, microglia can produce a variety of cytotoxic mediators, including nitric oxide (NO), proin- flammatory cytokines, excitatory amino acid, reactive oxygen intermediates, and arachidonic acid and derivatives [3]. Due to the mediating role of microglia, a great deal of effort has been made to develop pharmacological treatment of neurodegenerative diseases by targeting microglia and associative inflammatory factors [4]. In our recent explor- ation of new drugs, extracts of the traditional Chinese herb Tripterygium wilfordii Hook F (TWHF) have drawn our attention. The extract of TWHF has been reported to be effective in the treatment of a variety of inflammatory and autoimmune diseases, such as rheumatoid arthritis [5]. Triptolide (known as PG490) has been identified as the major component responsible for the immunosuppressive and anti-inflammatory effects of TWHF [6]. Accumulating data have demonstrated a strong anti-inflammatory action of PG490 on multiple tissues. However, mechanisms under- lying its anti-inflammatory action are poorly understood, especially its anti-inflammatory potential via a mechanism involving microglia. This study was therefore designed to evaluate effects of PG490 on microglia, particularly their production of several key inflammatory factors. In a primary microglia-enriched culture system that we developed previously from neonatal rats, effects of PG490 on LPS-stimulated NO, tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) release were tested. Putative effects of PG490 on inducible nitric oxide synthase (iNOS), TNF-a and IL-1b mRNA expression were then detected to define the PG490-sensitive regulation of these factors at the transcription level. MATERIALS AND METHODS Drugs and antibodies: PG490 was generously provided by Dr Jia-Run Zheng (Institute of Dermatology, Chinese Academy of Medical Sciences). The white crystal drug has a melting point of 226–2401C, and is 98% pure by reverse- phase high-pressure liquid chromatography evaluation. The mouse monoclonal antibody against the CR3 complement receptor (OX-42) was purchased from CHEMICON Inter- national, Inc. (Temecula, CA). Primary microglia-enriched cultures: Primary microglia were isolated and purified from whole brains of neonatal 1-day-old male Sprague–Dawley rats according to proce- dures described previously [7]. Briefly, once animals were deeply anesthetized, their brains were dissected. The 0959- 4965  c Lippincott Williams & Wilkins Vol14 No 7 23 May 2003 1091 GLIAL CELLS NEUROREPORT Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.