Xingshentongqiao decoction mediates proliferation, apoptosis, orexin-A receptor and orexin-B receptor messenger ribonucleic acid expression and represses mitogen-activated protein kinase signaling

Abstract

Background: Hypocretin (HCRT) signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ) is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R]) expression. The signaling pathways involved in these processes were also assessed.

Methods: The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK) pathway activation was performed to further assess the signaling mechanism of XSTQ.

Results: XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk) 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK).

Conclusions: XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.

Figure 1

Effects of Xingshentongqiao decoction (XSTQ) on proliferation of SH-SY5Y cells. (a) SH-SY5Y cell proliferation was determined by cell counting kit-8 assay after 0, 24, 48, 72, or 96 hours incubation without XSTQ (control) or with XSTQ at a concentration of 100, 200, 400, 600, 800, or 1000 μg/ml. Results are standardized to 1.0 at 0 hour. (b) The average proliferation of XSTQ-treated-SH-SY5Y cells is shown after 72 hours. Results are standardized to an average of 1.0 in the control. (c) Average proliferation of XSTQ-treated-SH-SY5Y cells is shown after 96 h. Results are standardized to an average of 1.0 in the control. Data represent the mean ± standard deviation of three independent experiments (**P < 0.01, *P < 0.05, vs. untreated control).

Figure 2

SH-SY5Y cell apoptosis in response to xingshentongqiao decoction (XSTQ) treatment. (a) Apoptosis was measured using an annexin V-fluorescein isothiocyanate apoptosis detection kit for SH-SY5Y cells incubated with XSTQ (0, 10, 100 or 1000 μg/mL) for 24 h. (b) The results from panel A were quantified for three independent experiments. The data are expressed as mean ± standard deviation (**P < 0.01 vs. untreated control).

Figure 3

Xingshentongqiao decoction (XSTQ) enhances orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) expression in SH-SY5Y cells. Induction of OX1R (a) and OX2R (b) was determined by quantitative real-time polymerase chain reaction at a range of times of exposure to 1000 μg/ml XSTQ. The data are expressed as mean ± standard deviation of OX1R or OX2R messenger ribonucleic acid (mRNA) expression relative to β-actin mRNA expression for three independent experiments and are standardized to 1.0 in the control sample (**P < 0.01, *P < 0.05 vs. untreated control).

Figure 4

Xingshentongqiao decoction (XSTQ) mediates dose-dependent downregulation of the phosphorylation of extracellular signal-regulated kinase (Erk1/2), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). (a) SH-SY5Y cells were incubated with XSTQ (0, 10, 100 or 1000 μg/ml) for 2 hours, and levels of phosphorylated Erk1/2, p38 MAPK and JNK were determined relative to total Erk1/2, p38 MAPK and JNK levels by western blotting analysis. (b-d) Levels of phosphorylated proteins were normalized to levels of total protein by densitometry. Data represent the mean ± standard deviation of three independent experiments and are standardized to 1.0 in the control (untreated) cells (**P < 0.01 vs. untreated control).

Figure 5

Xingshentongqiao decoction (XSTQ) mediates time-dependent downregulation of the phosphorylation of extracellular signal-regulated kinase (Erk1/2), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). (a) SH-SY5Y cells were incubated with 1000 μg/ml XSTQ for 0, 5, 15, 30, 60, 90, or 120 minutes, and levels of phosphorylated Erk1/2, p38 MAPK and JNK were determined relative to total Erk1/2, p38 MAPK and JNK by western blotting. (b-d) Levels of phosphorylated proteins were normalized to levels of total protein by densitometry. Data represent the mean ± standard deviation of three independent experiments and are standardized to 1.0 at time 0 (**P < 0.01, *P < 0.05, vs. untreated control).