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. 2011:2011:429384.
doi: 10.1093/ecam/nep025. Epub 2011 Jan 4.

San-Huang-Xie-Xin-Tang Protects against Activated Microglia- and 6-OHDA-Induced Toxicity in Neuronal SH-SY5Y Cells

Yu-Tzu Shih  1 , Ing-Jun Chen, Yang-Chang Wu, Yi-Ching Lo
Affiliations

San-Huang-Xie-Xin-Tang Protects against Activated Microglia- and 6-OHDA-Induced Toxicity in Neuronal SH-SY5Y Cells

Yu-Tzu Shih et al. Evid Based Complement Alternat Med. 2011.

Abstract

San-Huang-Xie-Xin-Tang (SHXT), composed of Coptidis rhizoma, Scutellariae radix and Rhei rhizoma, is a traditional Chinese herbal medicine used to treat gastritis, gastric bleeding and peptic ulcers. This study investigated the neuroprotective effects of SHXT on microglia-mediated neurotoxicity using co-cultured lipopolysaccharide (LPS)-activated microglia-like BV-2 cells with neuroblastoma SH-SY5Y cells. Effects of SHXT on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity were also examined in SH-SY5Y cells. Results indicated SHXT inhibited LPS-induced inflammation of BV-2 cells by downregulation of iNOS, NO, COX-2, PGE(2), gp91(phox), iROS, TNF-α, IL-1β, inhibition of IκBα degradation and upregulation of HO-1. In addition, SHXT increased cell viability and down regulated nNOS, COX-2 and gp91(phox) of SH-SY5Y cells co-cultured with LPS activated BV-2 cells. SHXT treatment increased cell viability and mitochondria membrane potential (MMP), decreased expression of nNOS, COX-2, gp91(phox) and iROS, and inhibited IκBα degradation in 6-OHDA-treated SH-SY5Y cells. SHXT also attenuated LPS activated BV-2 cells- and 6-OHDA-induced cell death in differentiated SH-SY5Y cells with db-cAMP. Furthermore, SHXT-inhibited nuclear translocation of p65 subunit of NF-κB in LPS treated BV-2 cells and 6-OHDA treated SH-SY5Y cells. In conclusion, SHXT showed protection from activated microglia- and 6-OHDA-induced neurotoxicity by attenuating inflammation and oxidative stress.

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Figures

Figure 1
Figure 1
Effects of SHXT on microglial BV-2 cells (a), neuroblastoma SH-SY5Y cells (b), lipopolysaccharide (LPS, 100 ng/mL) activated BV-2 treated non-differentiated (c) and differentiated (d) SH-SY5Y cells, and 6-hydroxydopamine (6-OHDA, 100 μM) treated non-differentiated (e) and differentiated (f) SH-SY5Y cells. Cells were treated with SHXT (25–200 μg/mL) for 24 h. Cell viability was determined by MTT assay. Bars represent the mean ± SEM from six independent experiments. # P <  .05 versus control, *P <  .05 versus LPS or 6-OHDA only.
Figure 2
Figure 2
Effects of SHXT on expression of iNOS, COX-2, gp91phox and HO-1 in BV-2 cells treated with lipopolysaccharide (LPS, 100 ng/mL) for 24 h. Cultures were pretreated with SHXT for 30 min before the addition of LPS treatment. Bars represent the mean ± SEM from six independent experiments. Densitometry analyses are presented as the relative ratio of protein/β-actin protein, and are represented as percentages of the control group. # P <  .05 versus control, *P <  .05 versus LPS only.
Figure 3
Figure 3
Effects of SHXT on the IκBα expression (a) and NF-κB activation (b) in lipopolysaccharide (LPS, 100 ng/mL)-treated BV-2 cells. Cultures were pretreated with SHXT for 30 min before the addition of LPS. Then the cells were collected at 1 h for NF-κB activity assay and at 24 h for IκBα protein analyses. Densitometry analyses are presented as the relative ratio of protein/β-actin protein, and they are represented as percentages of the control group. Changes of NF-κBp65 translocation levels are represented as percentages of the control group. Bars represent the mean ± SEM from six independent experiments. # P <  .05 versus control, *P <  .05 versus LPS only.
Figure 4
Figure 4
Inhibitory effects of SHXT on the expressions of nNOS (a), COX-2 (b) and gp91phox (c) in SH-SY5Y cells under co-culture with lipopolysaccharide (LPS, 100 ng/mL)-treated BV-2 cells. Cultures were pretreated with SHXT for 30 min followed by LPS treatment for 24 h. Densitometry analyses are presented as the relative ratio of protein/β-actin protein, and are represented as percentages of the control group. Bars represent the mean ± SEM from six independent experiments. # P <  .05 versus control, *P <  .05 versus LPS only.
Figure 5
Figure 5
Inhibitory effects of SHXT on the expressions of nNOS (a), COX-2 (b), IκBα(c) and NF-κB activation (d) on 6-hydroxydopamine (6-OHDA, 100 μM) treated SH-SY5Y cells. Cultures were pretreated with SHXT for 30 min before the addition of 6-OHDA. Then the cells were collected at 1.5 h for NF-κB activity assay and at 24 h for nNOS, COX-2 and IκBα protein analyses. Densitometry analyses are presented as the relative ratio of protein/β-actin protein, and are represented as percentages of the control group. Changes of NF-κB p65 translocation levels are represented as percentages of the control group. Bars represent the mean ± SEM from six independent experiments. # P <  .05 versus control, *P <  .05 versus 6-OHDA only.
Figure 6
Figure 6
Effects of SHXT on 6-hydroxydopamine (6-OHDA, 100 μM)-induced changes of gp91phox expression (a), iROS formation (b) and mitochondria membrane potential (c) in SH-SY5Y cells. The iROS was measured by using H2DCF-DA staining and mitochondrial membrane potential was measured by rhodamine 123. Densitometry analyses are presented as the relative ratio of protein/β-actin protein, and are represented as percentages of the control group. Bars represent the mean ± SEM from six independent experiments. # P <  .05 versus control group, *P <  .05 versus 6-OHDA only.
Figure 7
Figure 7
Hypothetical protective mechanisms of SHXT on microglia-mediated and 6-OHDA-induced toxicity in neuronal cells.
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