Effect of Angong Niuhuang Wan, cinnabar and realgar against lipopolysaccharide-mediated neurotoxicity / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To investigate the role of cinnabar and realgar in Angong Niuhuang Wan (AGNH) -produced neuroprotection against lipopolysaccharide ( LPS) -mediated neuronal damage and further explore the corresponding mechanisms. METHODS Primary rat midbrain neuron-glia cultures were used as an in vitro model to investigate effects of AGNH on LPS-mediated degeneration of dopamine (DA) neurons. The experiment was divided into normal control group, LPS model group, LPS + cinnabar (4 and 40 mg·L-1) groups, LPS + realgar (4 and 40 mg·L-1 ) groups and LPS + AGNH (40 and 400 mg·L-1 ) group. Drugs were added 30 min before LPS treatment. After 7 d, dopaminergic neurotoxicity was assessed through the quantification of tyrosine hydroxylase (TH)-positive neurons and morphological analysis of TH-positive neurons; the activation of microglia was evaluated using OX-42 antibody; the gene expression of tumor necrosis factor-α (TNF-α) and induced nitric oxide synthase (iNOS) mRNA in microglia was performed by real-time RT-PCR analysis, and the release of TNF-α and nitric oxide (NO) in the supernatant of neuron-glia cultures was determined respectively by the ELISA and Griess reagent. RESULTS Compared with normal control group, DA neurons in LPS model group decreased by 40% (P <0.05) , microglial activation was induced, the expression of TNF-α mRNA and iNOS mRNA in microglia increased 9 and 2 times, respectively ( P < 0. 05 ) , and subsequent production of TNF-α and NO in the supernatant of neuron-glia cultures increased 20 and 30 times, respectively (P<0.05). Compared with LPS model group, AGNH 400 mg·L-1 and realgar 40 mg·L-1 significantly attenuated LPS-mediated DA neuronal loss by 40% and 30% , respectively (P<0.05) and inhibited activation of microglia and expression of TNF-α mRNA by 61% and 52% (P <0.05). iNOS mRNA was reduced by 58% and 51% (P <0.05 ) in microglia. The subsequent release of TNF-α was reduced by 55% and 43% (P<0.05) and NO reduced by 53% and 34% (P<0.05) in the supernatant of neuron-glia cultures. Cinnabar had no inhibitory effect on LPS-induced changes. CONCLUSION AGNH protects LPS-induced neurotoxicity through its anti-inflammatory properties and realgar might be the key contributor to the neuroprotective action of AGNH, while cinnabar fails to show any neuroprotection.