Study On The Protective Effects And Mechanisms Of Tetrahydroxystilbene Glucoside On 6-OHDA-induced Dopaminergic Neuronal Damage

Objective: To investigate the protective effects of tetrahydroxystilbene glucoside(TSG) on 6-Hydroxydopamine(6-OHDA)-induced dopaminergic(DA) neuronal damage in rats. Methods: An in vivo Parkinson’s disease(PD) model was induced by direct injection of 6-OHDA into rat substantia nigra. Male Sprague–Dawley rats were randomly divided into control, TSG(50 mg/kg)alone, sham, 6-OHDA(8 μg), 6-OHDA +TSG(10 and 50 mg/kg) groups. Rats were pretreated with TSG for 30 min before 6-OHDA treatment and control groups were treated with saline. After 14 daily intraperitoneal injection of TSG, rat brains were removed, sectioned and processed for quantification of DA neurons by immunochemical staining with anti-tyrosine hydroxylase(TH) and OX-42(the specific marker of microglia) antibodies. In addition, primary rat neuron-glia co-cultures were prepared from the ventral mesencephalon tissues of embryonic day 14-15 rats. Cultures were randomly divided into control, TSG(80 μM) alone, 6-OHDA(40 μM) and 6-OHDA+TSG(20-80 μM) groups. Immunocytochemical staining was used to evaluate the DA neuronal damage with anti-TH antibody and the activation of microglia with anti-OX-42 antibody. Western blotting assay was applied to detect the protein expressions of TH and ionized calcium-binding adapter molecule-1(Iba-1), phosphorylated-ERK1/2 and phosphorylated- p38. The levels of nitric oxide(NO), TNF-α and IL-1β in the culture supernatants were measured with Griess Reagent and enzyme link immunosorbent assay(ELISA), respectively. Results: For in vivo experiment, compared with the control group, 6-OHDA induced DA neuronal damage and microglia activation in rat subtantia nigra. Compared with 6-OHDA group, TSG increased the DA neuronal number and inhibited microglia activation. In the neuron-glia co-culture, 6-OHDA induced DA neuronal damage and microglia activation and subsequently the release of NO, TNF-α and IL-1β in the culture medium. In the primary microglia culture, which was attenuated by TSG treatment. Moreover, TSG inhibited 6-OHDA-elicited protein expression of phosphorylated- ERK1/2 and p38. Conclusion: TSG protects DA neuronal damage against 6-OHDA-induced neurotoxicity and these neuroprotective effects are in part related to inhibiting the activation of microglia-mediated neuroinflammation.