Western blot analysis of (E) claudin-5, (F) zonula occludens 1 (ZO-1), and (G) intercellular adhesion molecule (ICAM)-1 levels in the ipsilateral hemisphere of sham-injured, vehicle-treated, and 10 mgkg1berberine-treated mice at 6 h, 12 h, and 1 day post-TBI

Western blot analysis of (E) claudin-5, (F) zonula occludens 1 (ZO-1), and (G) intercellular adhesion molecule (ICAM)-1 levels in the ipsilateral hemisphere of sham-injured, vehicle-treated, and 10 mgkg1berberine-treated mice at 6 h, 12 h, and 1 day post-TBI. up to day 28 post-injury. Berberine also reduced neuronal death, apoptosis, BBB permeability, and Rabbit polyclonal to SUMO3 brain edema at day 1 post-injury. These changes coincided with a marked reduction in leukocyte infiltration, microglial activation, matrix metalloproteinase-9 activity, and expression of inflammatory mediators. Berberine experienced no effect on Akt or Erk 1/2 phosphorylation. In mixed glial cultures, berberine reduced TLR4/MyD88/NF-B signaling. Berberine also attenuated neuronal death induced by microglial conditioned media; however, it did not directly protect cultured neurons subjected to stretch injury. Moreover, administration of berberine at 3 h post-injury also reduced TBI-induced neuronal damage, apoptosis and inflammationin vivo. Berberine reduces TBI-induced brain damage by limiting the production of inflammatory mediators by glial cells, rather than by a direct neuroprotective effect. == Introduction == Traumatic brain injury (TBI) triggers a series of neuroinflammatory processes that contribute to neuronal injury and failure of functional recovery . Post-traumatic inflammation is mediated by the activation of microglia and astrocytes and infiltration of circulating leucocytes into the affected area. Activated glia produce multiple pro-inflammatory mediators, including cytokines, chemokines, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Overproduction of these mediators is harmful to neighboring NSC-41589 neurons, which further activates glial cells and injures the remaining neurons through positive opinions[1],[2]. Neurotoxic proinflammatory cytokines can activate receptor-dependent apoptotic pathways via recruitment of adaptor molecules and caspase-8 or -10 activation[3],[4]. Thus, inhibition of glial activation and, therefore, production of inflammatory mediators may be a potential therapeutic strategy for protecting the damaged brain in TBI. Although a number of drugs targeting inflammatory pathways following TBI have been tested in clinical trials, none has conferred a significant benefit[5]. Berberine, an isoquinoline alkaloid isolated from medicinal natural herbs frequently used in traditional Eastern medicine, has multiple therapeutic effects for metabolic disorders, microbial contamination, neoplasms and inflammation[6]. Increasing interest has focused on its anti-inflammatory effects. In microglia, berberine suppresses neuroinflammatory responses[7],[8]and attenuates the production of inflammatory mediators through suppression of toll-like receptor 4 (TLR4)-nuclear factor-B (NF-B) signaling in animal models of endotoxemia[9],[10]. Substantial evidence also shows that berberine exerts neuroprotection in cerebral ischemia[11][15]and Alzheimer’s disease[16]. However, the therapeutic effect of berberine on TBI has yet to be evaluated, and the effective time at which berberine can be administered post-injury has not been investigated in preclinical studies as previous studies have administered it either prior to or shortly after onset[11][15]. The aim of the present study was to determine the effects of berberine in a mouse model of TBI. We also examined whether it attenuated TBI-induced glial activation, thereby preventing neuronal damage in both cell and animal models. == Material and Methods == == Animals == NSC-41589 Male C57BL/6J mice (age 810 weeks, excess weight 2328 g) were obtained from National Laboratory Animal Center (Taipei, Taiwan). All study protocols were approved by the Animal Research Committee at Cheng Hsin General Hospital (Animal permit number CHGH-97-02), and were conducted according to the Guideline for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996). The results of all studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals[17]. == Experimental protocol == All animals were randomized into one of the three following groups by using computer-generated random figures: (i) sham injury, (ii) controlled cortical impact (CCI) + vehicle, and (iii) CCI +10 mgkg1berberine. All NSC-41589 measurements explained below were also performed in a blinded manner. Berberine (Sigma, St. Louis, MO, USA) dissolved in 0.9% saline (0.2 mL) or a corresponding volume of vehicle (0.9% saline) was administered intraperitoneally 10 min following injury. The following measurements were assessed at the indicated time points following injury: 1) behavioral screening at days 1, 4, 7, 14, 21, and 28 (n = 12/group); 2) cresyl violet staining at 2 h, days 1 and 28 (n = 68 mice/group); 3) histology, brain water content, Evans blue (EB) dye extravasation, matrix metalloproteinase (MMP)-9 zymography, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA) at 6 h, 12 h, day 1 or day 4 (n = 57 mice/group); and 4) realtime quantitative reverse transcription polymerase chain reaction (RT-PCR) at 6 h (n = 7 mice/group). The dose and route of berberine were selected based on previous work in experimental cerebral ischemia[11],[14]and our pilot study in which concentrations of 5, 10, and 15 mgkg1were tested; both 10 mgkg1and 15 mgkg1improved behavioral deficits but there was no significant difference between the two groups (S1 Fig.). Another set of experiments was performed to investigate the delayed therapeutic potential of berberine for TBI. Berberine or vehicle was delivered intraperitoneally at 3 h post-injury, and the protective effects were assessed using cresyl violet staining, Fluoro-Jade B (FJB) histology, cleaved caspase-3 Western blot.