Effects of Four Compounds from Gentianella acuta (Michx.) Hulten on Hydrogen Peroxide-Induced Injury in H9c2 Cells

In previous studies, Gentianella acuta (Michx.) Hulten was reported to contain xanthones, iridoids, terpenoids, and sterols and is mainly used to cure hepatitis, jaundice, fever, headache, and angina pectoris. In this study, we used bioassay guided fractionation to identify compounds from G. acuta and investigated their activity against hydrogen peroxide (H2O2)-induced apoptosis of H9c2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic (GCLC) expression were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated using western blot. The results showed that all four compounds had protective effects on H9c2 cells. The transcription levels of HO-1 and GCLC significantly increased in H9c2 cells pretreated with norswertianolin (1), swetrianolin (2), demethylbellidifolin (3), and bellidifolin (4). However, compared to the model group, the transcription levels of Nrf2 were not enhanced by pretreatment with compounds 1, 2, and 4. The protein expression levels of HO-1 and GCLC in H9c2 cells were greater than that in the H2O2-treated group, and the expression of Nrf2 was not significantly changed except by swetrianolin treatment; inhibitors can reverse the protective effect by ZnPP (15 μM), BSO (10 μM), and brusatol (10 μM). The results indicated that the four compounds isolated from G. acuta inhibited the oxidative injury induced by H2O2 by activating the Nrf2/ARE pathway in H9c2 cells and provide evidence that G. acuta may be a potential therapeutic agent for the treatment of cardiovascular diseases.


Introduction
Gentianella acuta belongs to the family Gentianaceae and is also known by the Mongolian name Agute-Qiqige. It is an annual herbaceous plant, widely distributed to the north of China, the Mongolia Plateau, Siberia, and the Far East areas of Russia [1]. In previous studies, G. acuta was reported to contain xanthenes, iridoids, terpenoids, and sterols [2]. The whole grass is used as a Mongolian native medicine, mainly to treat hepatitis, jaundice, fever, and headache. A study showed that G. acuta had a significant effect on angina pectoris and other diseases [3].
Angina pectoris, the most common type of coronary heart disease, is a clinical syndrome caused by coronary insufficiency and acute and temporary myocardial ischemia [4]. Myocardial ischemia and reperfusion produce numerous reactive oxygen species (ROS) that can lead to oxidative injury of H9c2 cells, which is one of the main causes of H9c2 cells death after angina [5,6]. Hydrogen peroxide (H 2 O 2 ) is an important source of ROS, which directly oxidize lipids and proteins on the cell membrane, which then react with Fe 2+ to produce ⋅OH and other more active free radicals to induce apoptosis or necrosis. H 2 O 2 is one of the important causes of myocardial apoptosis through the induction of oxidative stress [7].
In this study, we used a bioassay guided method to isolate four compounds from G. acuta. We used chromatography to separate these active ingredients by analyzing the activity, discarding the inactive components, and repeating this process until the active ingredients were identified. We described the isolation and identification of these compounds, along with the evaluation of their inhibitory effects on H9c2 cells. To this end, we used a combination of high-performance liquid chromatography (HPLC), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and quantitative real-time polymerase chain reaction (qRT-PCR) methods to investigate the protective effects and preliminary mechanism of action of four compounds isolated from G. acuta. Furthermore, the effects on protein expression of relevant pathway molecules were evaluated using western blotting. The -butanol fraction (10 g) was chromatographed using a silica gel column (200-300 mesh, 200 g) to obtain four fractions (Frs. C1-4 [1.72, 5.7, 1.53, and 0.48 g, respectively]).

. . Identification of Antioxidant Compounds
. . . Cell Culture and Treatment. The H9c2 cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and a mixture of penicillin and streptomycin. The cells were maintained in a humidified incubator with an atmosphere of 5% CO 2 at 37 ∘ C.
. . . Determination of H O Model. We determined the appropriate concentration of H 2 O 2 using real-time cellular analysis (RTCA). H9c2 cells were seeded in a CIM-plate-16 microplate in a volume of 100 L and incubated for 12 h at 37 ∘ C in an atmosphere of 5% CO 2 . The H9c2 cells were divided into control and model groups. Then, 10 L H 2 O 2 (100, 200, 400, 600, and 800 mol⋅L −1 ) was added to the model groups, and the control groups were treated with 10 L DMEM complete medium, set up in three complex wells. The E-Plate 16 was replaced in the xCELLigence RTCA, monitored every 15 min, and recorded for 30 h. The effect of H 2 O 2 on the H9c2 cells was analyzed using the xCELLigence RTCA data analysis software. The cell CI value was used to determine the optimal H 2 O 2 concentration and duration of action.
. . . Cell Viability Assay. H9c2 cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well at a final volume of 100 L, and then they were divided into three groups: control, model, and treatment groups. Then, the treatment groups were treated with various concentrations of the crude extracts (0.4, 0.2, 0.1, 0.05, and 0.025 mg • mL −1 ) and the four monomeric compounds (50, 25, 12.5, 6.25, and 3.125 M) at five concentrations. After pretreatment for 12 h, 400 M H 2 O 2 solution was added to each well, followed by 10 L MTT solution for 4 h at 37 ∘ C, and then the supernatant was discarded and 150 L dimethyl sulfoxide (DMSO) was added to each well. The plates were oscillated at a low speed for 5 min at room temperature until all the crystals that had formed  . . HPLC Detection. The following chromatographic conditions were used: Waters Symmetry C18 column (4.6 mm × 150 mm, 5 m); mobile phase: acetonitrile-0.1% phosphoric acid water; flow rate, 1.0 mL • min −1 ; detection wavelength: 254 nm; injection volume, 10 L; and gradient elution, 55 min (Table 1) [8,9].
. . Western Blotting. The cells were washed with phosphatebuffered saline (PBS) and lysed on ice using radioimmunoprecipitation assay (RIPA, Beyotime, Jiangsu, China). The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer's instructions [11]. The samples were boiled at 98 ∘ C for 3 min, separated using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% nonfat dried milk for 60 min at room temperature. The blots were then incubated with the following rabbit polyclonal antibodies: anti-GAPDH (1:10000), anti-HO-1 (1:1000), anti-GCLC (1:1000), and anti-Nrf2 (1:1000). The blots were then washed extensively and incubated with horseradish peroxidase (HRP)-conjugated affinity pure goat anti-rabbit IgG (H+L, 1:5000). Visualization was performed using an enhanced chemiluminescence kit according to the instructions of the manufacturer.
. . Statistical Analysis. The data were analyzed using the statistical package for the social sciences (SPSS) 19.0, and the results are presented as the means ± standard deviation (SD) of three independent experiments. P-values < 0.05 were considered statistically significant. Figure 1, 600 and 800 mol⋅L −1 H 2 O 2 were too high to cause the appropriate level of cell death, whereas 200 mol⋅L −1 had little effects on the cells. Therefore, we determined the appropriate concentration of H 2 O 2 to be 400 M for mimicking oxidative stress-induced injury to H9c2 cells.

. . Crude Extracts Prevented H O -Induced Reduction in Cell
Viability. We used an MTT assay to analyze the effects of four crude extracts on H9c2 cells. As shown in Figure 2, the number of apoptotic cells significantly increased in the model group compared to the treatment group. Pretreatment with the four crude extracts for 12 h showed that the ethyl acetate fractions and -butanol extract significantly increased the percentage of viable H9c2 cells, suggesting that the two fractions protected H9c2 cells from apoptosis induced by Based on these results, we further evaluated the activity of seven subfractions (B1, B2-B10, B11-B14, C1, C2, C3, and C4) from the ethyl acetate and n-butanol fractions. The results show that B2-B10 and C2 significantly inhibited H 2 O 2induced apoptosis of H9c2 cells. The protective effects of B2-B10 and C2 are shown in Figure 3 Figure 5. We compared the plant contents of the four monomeric compounds using the area normalization method. These data indicated that there was a significant variation in the contents of the four monomeric compounds. The fingerprints of the four compounds and crude fractions are shown in Figure 6 and the relative contents are shown in Table 2.

. . Transcription Levels of HO-, GCLC, and Nrf .
We examined the transcription levels of HO-1, GCLC, and Nrf2 in H9c2 cells treated with compounds 1, 2, 3, and 4. As shown in Figure 7, the transcription levels of HO-1 and GCLC significantly increased in H9c2 cells pretreated with the indicated concentrations of compounds 1, 2, 3, and 4 for 12 h. Furthermore, the transcription levels of Nrf2 were increased in H9c2 cells pretreated with compound 3, whereas compounds 1, 2, and 4 did not alter the express compared with the model group.
. . Western Blot Analysis of HO-, GCLC, and Nrf . Figure 8 shows the expression of HO-1 and GCLC protein levels in H9c2 cells after treatment with 400 M H 2 O 2 , and the fluorescent intensities of HO-1 and GCLC in the treatment groups were greater than that in the H 2 O 2 -treated group (Figures 8(b) and 8(c)). However, none of the compounds except for swetrianolin significantly changed the expression of Nrf2 (Figure 8(d)). Pretreatment of H9c2 cells with ZnPP, BSO, and brusatol for 30 min and then addition of four compounds for 12 h, followed by exposure to H 2 O 2 , markedly decreased the cell viability, indicating that inhibitors can reverse the protective effects of four compounds (Figure 8(e)).

Discussion
In this study, we established a bioassay-guided method to detect the antioxidative activity of G. acuta and found that H 2 O 2 exposure significantly activated oxidative stress in H9c2 cells. This method can also be used to guide the more rapid and accurate separation of active ingredients than that achieved using conventional separation methods, which facilitates elucidation of the biological activity of medicinal plants, and is more conducive to the discovery of active or lead compounds. Therefore, the bioassay guided model combined with cell activity evaluation has important applicability in natural product research. Numerous studies have shown that the Nrf2/ARE pathway is critical to several antioxidant systems [12,13]. The transcription factor Nrf2 regulates the oxidative stress response in cells [14,15]. Nrf2 combines with Keap1 stably under normal physiological conditions. However, following activation, Nrf2 affects the expression of downstream antioxidant proteins such as GCLC and HO-1 to enhance the antioxidant capacity of the cells. Therefore, we choose the Nrf2/ARE pathway to elucidate the underlying protective mechanism in H9c2 cells.
We established a method to separate and analyze compounds 1, 2, 3, and 4 from G. acuta and conducted studies to validate the efficiency of the method. This specific model achieved good separation and the four compounds were successfully eluted from the G. acuta sample. The crude extract had protective effect on H9c2 cells from H 2 O 2 -induced apoptosis. The activity of the monomeric compounds obtained from the fractions was then tested. Results of the cytoprotective study showed that the chemical components in G. acuta markedly increased cell viability compared to H 2 O 2 alone.
In conclusion, the results of the present study demonstrated that four compounds are able to protect H9c2 cells from H 2 O 2 -induced damage, which may be associated with the activation of Nrf2/ARE pathway in H9c2 cells. These results provide evidence that G. acuta may be a potential therapeutic agent for the treatment of cardiovascular diseases.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.