Rhinacanthus nasutus protects cultured neuronal cells against hypoxia induced cell death

Abstract

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington's disease, Parkinson's disease and Alzeheimer's disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL⁻¹ root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL⁻¹ had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL⁻¹ reduced HT-22 cell proliferation. We also used H₂DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

Figure 1

The effect of R. nasutus in HT-22 cells measured using the MTT assay. ** ANOVA Dunnett’s multiple comparison Post hoc test compared to control P < 0.01. n = 6.

Figure 2

The Protective effect of R. nasutus against cell death induced by 18-hours hypoxia and 6-hours reoxygenation, measured using MTT assay. ^§§ One-way ANOVA Dunnett’s multiple comparison Post hoc test normoxia control vs. hypoxia control P < 0.01 n = 6. * ANOVA Dunnett’s multiple comparison Post hoc test compared to hypoxia control P < 0.05.

Figure 3

The protective effect of 24 h pretretment with R. nasutus against cell death caused by 18-hours hypoxia and 6-hours reoxygenation, measured using the Trypan blue exclusion assay. ^§§ One-way ANOVA Dunnett’s multiple comparison post hoc test normoxia control vs. hypoxia control P < 0.01. ** One-way ANOVA Dunnett’s multiple comparison post hoc test R. nasutus treated cells vs. hypoxia control P < 0.01 n = 3.

Figure 4

The protective effect of R. nasutus against against cell death caused by 18-hours hypoxia and 6-hours reoxygenation when added immediately before hypoxic condtions. Measured using the trypan blue exclusion assay. ^§§ One-way ANOVA Dunnett’s multiple comparison post hoc test normoxia control vs. hypoxia control P < 0.01. ** One-way ANOVA Dunnett’s multiple comparison post hoc test R. nasutus treated cells vs. hypoxia control P value < 0.01 n = 3.

Figure 5

The protective effects of R. nautus against cell death caused by 18-hour hypoxia and 6-hours reoxygenation. HT-22 Cell cultured in 6 well culture plates at a density of 10⁵ cells per well and photographed using a soligor microscope adaptor tube for Canon A650 with the Austria micros microscope 10 × objective and 6 × zoom on the camera. (A). HT-22 cells subjected to normoxic conditions cells; (B). Untreated HT-22 cells subjected to hypoxia 18-hours, reoxygenation 6-hours; (C). HT-22 cells pre treated with 10 µg mL⁻¹ R. nasutus subjected to 18-hours of hypoxia and 6-hours of reoxygenation; (D). HT-22 Cells pretreated with 1 µg mL⁻¹ R. nasutus subjected to 18-hours of hypoxia and 6-hours of reoxygenation; (E). HT-22 cells treated with 10 µg mL⁻¹ R. nasutus at the time of being subjected to 18-hours of hypoxia and 6-hours of reoxygenation; (F). HT-22 cells treated with 1 µg mL⁻¹ R. nasutus at the time of being subjected to 18-hours of hypoxia and 6-hours of reoxygenation.

Figure 6

LDH release from HT-22 resulting from being subjected to 18 hours of hypoxia and 6-hours of reoxygenation (A) (n = 5) or 24-hours rexoygenation (B) (n = 3) * ANOVA Dunnett’s post hoc test P < 0.05. ** ANOVA Dunnett’s post hoc test P < 0.01.

Figure 7

Carboxy-H₂-DCFDA assay. HT-22 cells were cultured in 96 well plates at 10⁴ per well and treated with R. nasutus root ethanol extract (0.1–10 µg mL⁻¹) over night before staining for reactive oxygen species with carboxy-H₂-DCFDA, and the emission read at 521 nm after excitation at 494 nm. The data was normalized to the % fluorescence of the untreated hypoxia cells. * ANOVA Dunnett’s post hoc test P < 0.05. ** ANOVA Dunnett’s post hoc test P < 0.01. ^§§ ANOVA Dunnett’s post hoc normoxia control vs. hypoxia control test P < 0.01.

Figure 8

(A) Equipment used to generate hypoxic conditions. These include airtight lockable container with compartments for water, the anearopack and culture plates, the anaeropack from Mitsubishish Gas Company and a 96 well tissue culture plate; (B) Oxygen indicator tab in side airtight container with cells.

Figure 9

(A) Oxygen indicator tab in hypoxic conditions; (B) Oxygen indicator tab in normoxic conditions.