Yanang water extract exhibits a protective effect against methomyl-induced cytotoxicity in RAW 264.7 cells via suppression of apoptosis and cell cycle arrest

2.1 Preparation of Yanang extract

The leaves of Yanang (Tiliacora triandra) were gathered from Khon Kaen province in the Northeastern region of Thailand. The leaves were washed under a tap, cut into small pieces, and ground with distilled water at a 1:8% w/v ratio. The extract was then filtered through cotton cloth. The filtrate was centrifuged at 6,068 ×g at 4 °C for 30 min. The pellet was discarded, and the supernatant was collected as Yanang water extract (YWE). The protein concentration and total phenolic content (TPC) were measured. The extract was kept cold (−20 °C) until further use in subsequent experiments.

2.2 Protein concentration measurement

The Bradford assay was employed to assess the concentration of protein (Bradford, 1976). Briefly, 1,000 µL of Bradford dye was mixed with 1 µL of YWE and incubated for 10 min. The absorbance was recorded at 595 nm (Microplate reader, Varioskan LUX, USA). The concentration of protein was subsequently determined using the BSA standard curve.

2.3 Total phenolic content (TPC) measurement

The TPC of YWE was assessed using the Folin-Ciocalteu (FC) reagent (Singthong et al., 2014). Briefly, 1,000 µL of YWE was mixed with 100 µL of FC reagent, 300 µL of 20 % sodium carbonate, and 500 µl of distilled water. The reaction mixture was incubated for 2 h at 50 °C, and a microplate reader (Varioskan LUX, USA) was used to record the absorbance at 765 nm. A calibration curve of standard gallic acid was used to convert the measured absorbance values into gallic acid equivalents (µg GAE/mL), which served as a measure of the total phenolic content.

2.4 Cell viability / cytotoxicity assay

RAW 264.7 cell viability (ATCC, Manassas, VA, USA) was evaluated using the MTT assay. In a 96-well plate, the cells (2.5 × 10⁴ cells/well) were cultured for 24 h at 37 °C with 5 % CO₂ in DMEM (Dulbecco's Modified Eagle Medium). Afterward, cells were exposed to YWE for an additional 24 h at doses ranging from 2.5 – 10 µg/mL. To assess the protective effect of YWE against methomyl-induced cytotoxicity in RAW 264.7, the cells were co-treated with 11,000 µM methomyl and 2.5 – 10 µg/mL of YWE for 24 h. Subsequently, the medium was substituted with 100 μL of MTT solution (0.5 mg/mL) and incubated for 30 min at 37 °C with 5 % CO₂. The purple crystal (formazan) was then solubilized in 100 μL of DMSO, and the absorbance at 570 nm was quantified using a microplate reader (Varioskan LUX, USA). To calculate cell viability (%), the following formula was used: $$\%Cellviability=\left(A{570}_{\mathit{test}}/A{570}_{\mathit{control}}\right)\times 100$$ where A570_test refers to the absorbance of cells treated with either YWE, methomyl, or co-treatment at 570 nm. A570_control refers to the absorbance of untreated cells at 570 nm.

2.5 Dual fluorescent staining apoptosis assay

Apoptosis was studied using the dual staining approach with acridine orange/ethidium bromide (AO/EB). RAW 264.7 cells (1.0 × 10⁵ cells/well) were plated into a 48-well plate and cultured for 24 h at 37 °C with 5 % CO₂. After that, cells were incubated with 11,000 µM methomyl and supplemented with YWE (2.5 – 10 µg/mL) for an additional 24 h. After trypsinization, cells were resuspended in a fresh medium and centrifuged at 95 ×g for 5 min. Following this, a dual staining solution consisting of 10 mg/mL AO and 10 mg/mL EB in PBS was applied to stain the cell pellet in a 1:1 ratio. The cells were incubated in the dark for 15 min. A 10 μL aliquot of the stained cell suspension was transferred to microscope slides, coverslipped, and observed under a fluorescence microscope (Carl Zeiss Microscopy, USA) for analysis of apoptotic morphology.

2.6 Flow cytometry-based apoptosis assay

After being plated into a 12-well plate, RAW 264.7 cells (1.5 × 10⁵ cells/well) were cultured for 24 h at 37 °C with 5 % CO₂. After that, cells were exposed to 11,000 µM methomyl and supplemented with YWE (2.5 – 10 μg/mL) for 24 h. The cells were then trypsinized, suspended in fresh DMEM, and centrifuged at 95 ×g for 5 min. After rinsing the cell pellet with ice-cold PBS, cells were re-suspended in an appropriate volume of 1 × Annexin V binding buffer (BioLegend, USA) to achieve 1 × 10⁶ cells/mL. Annexin V-FITC (5 μL) and propidium iodide (PI) (10 μL) (BioLegend, USA) were then added to 100 μL of this cell suspension. 400 μL of binding buffer was added after the mixture was left in the dark for 15 min. Apoptosis rates were analyzed using a flow cytometer (BD FACSCanto II, BD Biosciences, USA) with BD Accuri C6 software for data interpretation.

2.7 Flow cytometry-based cycle arrest assay

The cell cycle stage was assessed using PI staining. After treatment of RAW 264.7 cells with 11,000 µM methomyl and various concentrations of YWE, as described earlier, cell pellets were collected, rinsed with ice-cold PBS, and fixed with 400 μL of ice-cold 70 % ethanol at 4 °C overnight. The cells were then centrifuged at 95 ×g for 5 min and re-suspended in 200 μL of PBS. 100 μL of cell suspension was transferred into a flow tube and incubated in the dark with 2.5 μL of RNase A (20 mg/mL) and 2 μL of PI (1 mg/mL) for 30 min. After that, 400 μL of PBS was added to the cell suspension, and a Flow Cytometer (BD FACSCanto II, BD Biosciences, USA) was employed to assess the cell cycle.

2.8 Gene expressions study using real-time PCR

After RAW 264.7 cells were cultured and treated with 11,000 µM methomyl along with various concentrations of YWE, as described earlier, the cell pellets were harvested and processed for RNA extraction using Trizol reagent (Invitrogen, USA). The cDNA was synthesized according to the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc, USA). The PCR mixture included 2 μL of cDNA, 10 μL of LightCycle® SYBR® Green I Master mix (Roche, Switzerland), and 1 μL of each primer (10 μM) in a total volume of 20 μL. All PCR reactions were amplified using the LightCycler 480 instrument (Roche, Switzerland). The annealing temperatures for each gene-specific primer were determined individually as listed in Table 1. The comparative 2^-ΔΔCT approach was employed to determine the relative alterations in gene expression levels. The GAPDH gene was used to normalize target gene expression levels.

2.9 Proteomics analysis using liquid chromatography mass spectrometry

RAW 264.7 cells were cultured and treated with 11,000 µM methomyl and various concentrations of YWE as described earlier. Then, cells were harvested, and proteins were extracted using 50 mM Tris-HCl pH 7.0 and 0.5 % SDS, with sonication at amplitude levels of 80 % for 3 s. Centrifugation was performed at 9,481 ×g for 15 min and the supernatant was used for protein extraction by incubating with ice-cold acetone containing 0.1 mM DTT (1:5 v/v ratios) for 16 h at −20 °C. The protein was subsequently digested with trypsin at a ratio of 1:20 before being incubated at 37 °C for 6 h. Peptides were fractionated by ultra-performance liquid chromatography (UPLC) and C₁₈-reverse phase chromatography. Quantitative data analysis was processed by Decyder Ms 2.0. Protein identification was conducted using Mascot. To visualize all possible intersections among proteome datasets, a Venn diagram was generated using the jvenn tool online.

2.10 Statistical methods

Data from three independent experiments are shown as means ± SD. SPSS 20 software was used to conduct the statistical analysis. Duncan's multiple range test was employed for post-hoc comparisons after one-way ANOVA was used to evaluate differences between the treatment and control groups. A 0.05 threshold for statistical significance was applied.

3.1 Protective effect of YWE against methomyl-induced cytotoxicity in RAW 264.7 cells

The YWE was analyzed for total protein and total phenolic content using the Bradford assay and the Folin-Ciocalteu method, respectively. The results indicate that YWE showed protein and total phenolic content at 7.42 ± 0.24 µg/mL and 5.23 ± 0.41 µg GAE/mL, respectively. Rapid protein concentration determination with the Bradford method was used for estimating YWE concentration in all experiments. MTT assay confirmed that YWE (2.5–10 µg/mL) exhibited no cytotoxicity in RAW 264.7 cells (99.08 – 100.78 % cell viability; Fig. 1A). These non-toxic YWE concentrations were then tested for protection against methomyl-induced cytotoxicity. In Fig. 1B, it is evident that methomyl at a concentration of 11,000 μM significantly induced cytotoxicity in RAW 264.7 cells, resulting in a viability of 48.16 %. However, co-treatment with YWE at concentrations of 2.5, 5, and 10 µg/mL significantly reduced this effect, with cell viabilities of 57.13 %, 60.86 %, and 64.40 %, respectively (Fig. 1B).

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Fig. 1

Toxicity and protective effect of Yanang water extract (YWE) on methomyl- induced cytotoxicity in RAW 264.7 cells. (A) RAW 264.7 cells were exposed to YWE at concentrations of 2.5 – 10 µg/mL for 24 h and then assessed using the MTT assay to determine their effect on cell viability. (B) The RAW 264.7 cells viability was measured using MTT assay after a 24 h incubation with 11,000 µM methomyl and supplementation with different concentrations of YWE (2.5 – 10 μg/mL) to evaluate its protective effect. The data are presented as mean ± SD. Different letters on the top of each bar are significantly different, p ≤ 0.05.

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3.2 Protective effect of YWE on methomyl-induced apoptosis in RAW 264.7 cells

The morphological apoptotic changes of RAW 264.7 cells with AO/EB dual staining are shown in Fig. 2A. AO stains live cells green, while EB stains apoptotic cells orange or red. The untreated control group showed predominantly green live cells, whereas the methomyl-treated group exhibited more apoptotic cells. Co-treatment with YWE (2.5 – 10 μg/mL) led to a concentration-dependent reduction in apoptotic cells.

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Fig. 2

Protective effects of Yanang water extract (YWE) on methomyl-induced apoptosis in RAW 264.7 cells. After treatment with methomyl and different concentrations of YWE (2.5 – 10 μg/mL) for 24 h, RAW 264.7 cells were stained with AO/EB and observed under fluorescence microscopy (20×) (A). The cells undergoing apoptosis were further measured by annexin V-FITC/PI flow cytometry assay and the apoptosis rate was determined and represented as a bar graph (B). The protective effect of YWE on apoptosis related genes, Caspase-9 (C) and Caspase-3 (D) was assessed through real-time PCR. The data are presented as mean ± SD. Different letters on the top of each bar are significantly different, p ≤ 0.05.

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Annexin V-FITC/PI flow cytometry confirmed methomyl-induced apoptosis in RAW 264.7 cells, which was significantly reduced with YWE treatment. Apoptotic cell percentages decreased in a concentration-dependent manner to 95.54 %, 71.26 %, and 57.87 % with 2.5, 5, and 10 μg/mL of YWE, respectively (Fig. 2B).

Quantitative real-time PCR analysis revealed that methomyl treatment increased the expression of Caspase-9 and Caspase-3, key regulators of apoptosis, compared to the untreated group. Conversely, YWE co-treatment (2.5 – 10 µg/mL) downregulated these genes, suggesting that YWE may exert its protective effect against methomyl-induced apoptosis by regulating caspase expression (Fig. 2C, 2D).

3.3 Protective effects of YWE on cell cycle arrest induced by methomyl toward RAW 264.7 cells

DNA flow cytometric analysis indicated that methomyl significantly induced G₂/M cell cycle arrest by increasing the G₂/M phase from untreated control to 7.30 % ± 0.28. However, the percentages of RAW 264.7 cells in the G2/M phase were significantly reduced by co-treatment with YWE (2.5, 5, and 10 μg/mL) to 6.45 % ± 0.49, 5.85 % ± 0.30, and 3.30 % ± 0.18, respectively. These findings suggest that YWE exerts protective effects by preventing methomyl-induced cell cycle arrest in RAW 264.7 cells, as evidenced by the reduced G2/M phase population (Fig. 3).

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Fig. 3

Protective effect of Yanang water extract (YWE) on methomyl-induced cell cycle arrest in RAW 264.7 cells. After treatment with methomyl and different concentrations of YWE (2.5 – 10 μg/mL) for 24 h, RAW 264.7 cells were stained with PI and measured by flow cytometry. The percentage of RAW 264.7 cells in G₂/M peak is presented as mean ± SD. Different letters on the top of each bar are significantly different, p ≤ 0.05.

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3.4 Protective effects of YWE on the protein patterns in methomyl-induced toxicity in RAW 264.7 cells

The proteomic analysis revealed 681 proteins expressed in RAW 264.7 cells under 5 different conditions, including untreated control, methomyl alone, and co-treatment of methomyl with 2.5, 5, and 10 µg/mL of YWE. The number of expressed proteins in each group was 550, 536, 575, 565, and 561, respectively (Fig. 4). A Venn diagram of differentially expressed proteins (DEPs) indicated that 423 proteins were shared commonly among all 5 conditions, while 7 proteins were only observed in the untreated control and methomyl treated groups, 5, 8 and 1 proteins were only observed in co-treatment of methomyl with 2.5, 5, and 10 μg/mL of YWE, respectively (Table S1). The functional proteins related to cell death and cell cycle pathway are indicated in Table 2-4. Table 2 lists the proteins exclusively observed in each group, which have known functions in apoptosis and cell cycle pathways. Of these, 4 proteins were only found in the methomyl treated group, including RAC-alpha serine/threonine-protein kinas (AKT1), Gamma-aminobutyric acid receptor subunit alpha-3 (GBRA3), LAG1 longevity assurance homolog 1 (LASS1) and programmed cell death protein 2-like (PDD2L). Meanwhile, co-treatment with 2.5 – 10 μg/mL of YWE altered functional proteins by promoting an expression of CUB and sushi domain-containing protein 1 (CSMD1) at a dose of 2.5 μg/mL, Homeobox protein DLX-6 (DLX6), Spectrin alpha chain (SPTA1) and E3 ubiquitin-protein ligase TRIP12 (TRIPC) at a dose of 5 μg/mL, and RING finger protein 113A (R113A) at a dose of 10 μg/mL.

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Fig. 4

Venn diagram presenting the expressed proteins of 5 treatments, including untreated control (550 proteins), methomyl treatment (536 proteins), and supplementation of 2.5 μg/mL Yanang water extract (YWE) (575 proteins), 5 μg/mL YWE (565 proteins), and 10 μg/mL YWE (561 proteins) to methomyl treated RAW 264.7 cells.

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Moreover, 39 proteins were differentially expressed across the groups of methomyl treatment, and co-treatments of methomyl with YWE were also evaluated (Table S2). Of these, the expression levels of 2 apoptosis-related proteins, Death-inducer obliterator 1 (DIDO1) and Cullin-5 (CUL5) were found to have a marked decrease in a dose-dependent manner of YWE (Table 3).

Among the 423 proteins commonly identified in all 5 conditions (Table S3), 7 proteins are involved in apoptosis and cell cycle arrest. These include DNA replication licensing factor MCM5 (MCM5), G2/mitotic-specific cyclin-B2 (CCNB2), Ras GTPase-activating-like protein IQGAP1 (IQGA1), Retinoblastoma-binding protein 6 (RBBP6), Tyrosine-protein kinase JAK2 (JAK2), Storkhead-box protein 2 (STOX2), and PRKC apoptosis WT1 regulator protein (PAWR). Their expression levels exhibited slight changes, as shown in Table 4.