Buyang Huanwu Tang (补阳还五汤) protects H2O2-induced RGC-5 cell against oxidative stress and apoptosis via reactive oxygen species-mitogen-activated protein kinase signaling pathway

doi:10.19852/j.cnki.jtcm.2022.06.004

Abstract

Abstract:

OBJECTIVE: To study the molecular mechanism of Buyang Huanwu Tang(补阳还五汤) (BHT) protecting retinal ganglion cells (RGCs) from oxygen induced oxidative stress and apoptosis after anterior ischemia.

METHODS: In this study, the Chinese herbs of BHT were extracted by first boiling in water, then were filtered, concentrated, and freeze-dried. The chemical profile of BHT extract was determined by liquid chromatography mass spectrometry (LC-MS). H₂O₂-induced RGC-5 cells were used as a cell model to investigate the protective effect and mechanism of BHT on RGCs.

RESULTS: The survival rate of damaged RGC-5 by BHT was significantly increased by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazolium-romid method. Fluorescence activating cell sorter (FACS analysis) showed that BHT could significantly reduce apoptosis induced by oxidative stress via the reactive oxygen species (ROS)-mitogen-activated protein kinase (MAPK)-Caspase-3 signal pathway.

CONCLUSION: BHT possesses a high antioxidant capacity and could significantly reduce ROS levels of RGC-5 cells damaged by H₂O₂.Therefore, the present study has provided possible alternative strategies for the prevention and treatment of ischemic optic disease by using traditional Chinese herbal formulas.

Key words: optic neuropathy, ischemic, oxidative stress, apoptosis, reactive oxygen species, mitogen-activated protein kinase, caspase 3, Buyang Huanwu Tang

ZHENG Wei, WANG Mingxing, LIU Shanxue, LUAN Chao, ZHANG Yanqiu, XU Duoduo, WANG Jian. Buyang Huanwu Tang (补阳还五汤) protects H₂O₂-induced RGC-5 cell against oxidative stress and apoptosis via reactive oxygen species-mitogen-activated protein kinase signaling pathway[J]. Journal of Traditional Chinese Medicine, 2022, 42(6): 885-891.

Figures/Tables 6

Table 1 Results of components in Buyang Huanwu Tang by mass spectrometry

Deduced component name	Retention time (min)	Mass spectrum fragment	Standard molecular weight	Standard molecular weight
Caffeic acid	3.74	[M-H]^-	180	180
Chlorogenic acid	3.03	[M+H]⁺	354	354
Paeoniflorin	5.82	[M+H]⁺, [M+Na]⁺, [M+K]⁺	502	502
Ferulic acid	6.98	[M+H]⁺	194	194
Astragaloside	34.39	[M+H]⁺, [M+Na]⁺, [M+K]⁺	806	806

Figure 1 The ion current diagram of references A: Caffeic acid; B: Chlorogenic acid; C: Paeoniflorin; D: Ferulic acid; E: Astragaloside.

Figure 2 The mass spectrum of each peak in ion current diagram of BHT and the deduced structure formul A: Caffeic acid; B: Ferulic acid; C: Paeoniflorin; D: Chlorogenic acid; E: Astragaloside; BHT: Buyang Huanwu Tang.

Figure 3 Effects of H2O2 and BHT on cell viability. A: cells under the microscope (40 x) (H2O2 in a concentration ranging from 25 to 200 μM for 24 h, BHT concentration for for 24 h); B: MTT assay; C: Cell apoptosis assay; D: The protective effects of BHT. Pretreatment with BHT (range from 12.5 to 100 μg/mL) for 24 h prior to 100 μM H2O2. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Values are expressed as mean ± SD, aP < 0.05, bP < 0.01 (Model vs Blank). BHT: Buyang Huanwu Tang; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA: Analysis of Variance.

Figure 4 Effect of BHT on oxidative stress and inflammation in RGC-5 cells.. A-D: cytokines release assay (RGC-5 cells were pretreated with BHT (25 μg/mL) for 24 h, followed by stimulation of 100 μM H2O2 for 6 h); E, F: intracellular ROS generation; G-I: expression levels of nuclear factor kappa-B (NF-κB), (inhibitor of NF-κB) IκBα and Tumor Necrosis Factor-α (TNF-α); J: EGF in RGC-5 cells. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Values are expressed as mean ± SD, aP < 0.01, d P < 0.05 (Model vs Blank) indicates a significant difference from the blank group. b P < 0.01, c P < 0.05, indicates a significant difference from the model group (BHT, NAC vs Model).

Figure 5 BHT protect RGC-5 cells from apoptosis. A, B: Typical histogram of RGC-5 apoptosis ratio (RGC-5 cells were pretreated with BHT (25 μg/mL) for 24 h, followed by stimulation of 100 μM H2O2 for 6 h); C-G: The expression of p38 MAPK and apoptotic related proteins Bcl-2, Bax and Caspase-3. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Values are expressed as mean ± SD, #P < 0.05, ##P < 0.01 indicates a significant difference from the blank group (Model vs Blank). *P < 0.05, **P < 0.01 indicates a significant difference from the model group (BHT, NAC vs Model).

References 14

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Buyang Huanwu Tang (补阳还五汤) protects H₂O₂-induced RGC-5 cell against oxidative stress and apoptosis via reactive oxygen species-mitogen-activated protein kinase signaling pathway

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