Icaritin protects SH-SY5Y cells transfected with TDP-43 by alleviating mitochondrial damage and oxidative stress

Reagents

Reagent-grade ICT, (purity 99.5% by HPLC analysis) was obtained from Zhongke Quality Inspection Biotechnology Co., Ltd. (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO in the medium was less than 0.1% (v/v). Human TDP-43 and polybrene were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China), a TransZol Up Plus RNA Kit and EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix were purchased from TransGen Biotechnology Co., Ltd. (Beijing, China) and NuHi Robustic SYBR Green Mix was purchased from Nuhigh Biotechnologies Co., Ltd. (Suzhou, China). High-Sig ECL Western Blotting Substrate was purchased from Tanon Technology Co., Ltd. (Shanghai, China). RIPA buffer (high) (R0010) was purchased from Solarbio Life Science (Beijing, China). A GAPDH antibody, a TDP-43 antibody, a CytC antibody, and HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) were purchased from Proteintech Group (Wuhan, China). A goat anti-mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific (Waltham, USA).

Cell culture

SH-SY5Y human neuroblastoma cells were purchased from Wuhan University Collection Center. The cells were cultured in DMEM-F12 medium containing a mixture of 15% fetal bovine serum and 1% penicillin streptomycin in a 5% CO₂ cell culture incubator at 37 °C. When the cells covered approximately 80% of the bottom of the bottle, they were collected in a single tube and centrifuged at 1000 rpm for 5 min after trypsin digestion. After discarding the supernatant, the cells were seeded in 96-well plates, 24-well plates, or 6-well plates at a density of 5  × 10⁴/mL in complete medium, and the medium was changed every 2 days.

Determination of the optimal multiplicity of infection (MOI)

Virus transduction construct, cloning, and production were carried out by Hanbio Biotechnology Co., Ltd. The detailed steps and more information are provided in the supplementary files. SH-SY5Y cells showing good growth were chosen. The SH-SY5Y cells (1 ×10⁵ cells/mL) were plated in a 96-well plate at a density of 1 ×10⁴ cells per well and maintained at 37 °C and 5% CO₂ A total of 200 µL of fresh medium containing virus at an MOI of 0, 10, 20, 30, 40, 50, or 60 (including 1.2 µL of polybrene at a working concentration of 6 µg/mL). After 12 h, the transfection was terminated, and the optimal MOI was selected by observing and photographing the cells under a fluorescence microscope.

Cell viability determination

Cell viability was assessed by the CCK-8 assay (Apexbio, USA); in this assay, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt produces water-soluble maz dye after it is formed and undergoes biological reduction in the presence of electron carriers. Briefly, SH-SY5Y cells (1 ×10⁵ cells/mL) were seeded in each well of a 96-well plate for 24 h. After treatment, CCK-8 solution (10 µL) was added to each well of the 96-well plate, the cells were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm with a Synergy HTX microplate reader (Bio Tek, USA).

Real-time fluorescent quantitative PCR (qPCR)

TransZol Up reagent was used to extract total RNA. For first-strand cDNA synthesis and gDNA removal, RNA template and primers (Table 1) were designed by Sangon Biotech Co., Ltd. (Shanghai, China), mixed with RNase-free water, incubated at 65 °C for 5 min, incubated on ice for 2 min, and then added to other reaction components (Table 2). qPCR was performed on a t100 thermal cycler/621 BR 18022 gradient PCR instrument (Bole Biotechnology Co., Ltd.) using NuHi Robustic SYBR Green Mix (Suzhou Nuhigh Biotechnologies Co., Ltd.). Each reaction (25 µL volume) consisted of 1.5 µL cDNA, 12.5 µL NuHi Robustic SYBR Green Mix, 0.5 µL forward and reverse primers (10 µM) and 10 µL RNase-free double distilled water. The qPCR conditions were as follows: initial denaturation at 95 °C for 2 min, 40–50 cycles at 95 °C for 5 s, and 55 °C for 25 s. Melting curve analysis was performed from 55 °C to 95 °C to determine the specificity of the qPCR primers.

Western blot assay

Cells were collected, lysed with RIPA lysis buffer, and then centrifuged at 12,000 rpm at 4 °C for 15 min. The total protein concentration was measured with a BCA protein assay kit (Beijing Solarbio). The proteins were then heated at 100  °C for 5 min for denaturation. Equal amounts of total protein (25 µg per lane) were loaded on 12% SDS-PAGE gels and separated. After sample loading, the voltage was set to 90 V and then to 120 V until the sample reached the bottom of the gel. The proteins were then transferred to a nitrocellulose (NC) film by the sandwich method. The membrane was washed three times with TBST and then blocked with 5% skimmed milk. The membrane was then incubated for 12 h at 4 °C with a GAPDH antibody (1:1000; Proteintech Group), TDP-43 antibody (1:1000; Proteintech Group), and CytC antibody (1:1000; Proteintech Group). The membrane was washed with TBST 3 times and incubated with HRP-conjugated Affinipure goat anti-rabbit lgG (H+L) (1:1000; Proteintech Group) for 2 h at room temperature. The membranes were developed using hydrogen peroxide and Supersignal West Pico Luminol (Pierce, Seymour Fisher Technologies). Finally, High-Sig ECL Western Blotting Substrate (Shanghai Tanon Technology Co., Ltd.) was used to visualize the membrane.

JC-1 assay

A JC-1 MMP test kit (Shanghai Yeasen Biotechnology Co., Ltd.) was used according to the manufacturer’s protocol. The culture medium of each group of cells was discarded, the cells were wash once with PBS, and the same amount of culture medium and JC-1 staining working solution was added and mixed well. Afterwards, the cells were placed in a 37 °C cell incubator and incubated for 20 min. Then, the supernatant was discarded, the cells were washed twice with prepared 1 × JC-1 staining buffer, and cell culture medium was added. After this, the cells were washed with PBS and observed under an inverted fluorescence microscope (IX73; Olympus, Japan).

Changes in ATP content, T-AOC, GSH-Px activity, total SOD (T-SOD) activity, copper/zinc SOD (CuZn-SOD) activity, manganese SOD (Mn-SOD) activity and MDA content

We used a commercial ATP content test kit, GSH-Px test kit, SOD typing test kit, MDA test kit (Nanjing Jiancheng Biological Engineering Institute) and T-AOC test kit (Shanghai Beyotime Biotechnology Co., Ltd.). A BCA protein quantitative kit (Beijing Solarbio) was used to determine the protein content.

To measure ATP levels, reagents and the sample to be tested were added to tubes according to the manufacturer’s protocol. A blank tube, a standard tube, experimental tubes, and a control tube were analyzed, and the experiment was performed in triplicate. The absorbance value of each tube at a wavelength of 636 nm was measured (Multiskan GO, Thermo Scientific, USA).

To determine T-AOC, cells were scraped off, lysed on ice, and centrifuged at 4 °C at 12,000 rpm in a low-temperature centrifuge for 5 min, and the supernatant was taken for determination. Reagents and the sample to be tested were added to each well according to the manufacturer’s instructions, the absorbance value of each well at a wavelength of 734 nm was measured, and the T-AOC of the sample was calculated according to the standard curve.

To measure GSH-Px levels, prepared cell homogenates were centrifuged in a low-temperature centrifuge at 2000 rpm for 10 min, and the supernatant was centrifuged at 10,000 rpm in a low-temperature centrifuge for 15 min. After adding 0.5 mL of PBS and mixing, the prepared detection reagents were added, and the absorbance value of each sample was determined at a wavelength of 412 nm.

To analyze SOD levels, T-SOD, CuZn-SOD, and Mn-SOD activity in SH-SY5Y cells was measured using a SOD typing assay kit via the hydroxylamine method according to the manufacturer’s protocol. SH-SY5Y cells were seeded into a 6-well plate, and the absorbance of the samples was measured at 550 nm using a microplate reader.

To assess MDA levels, treated cells were washed with cold PBS and homogenized in PBS in a glass homogenizer to form a suspension. The supernatant was used for analysis according to the manufacturer’s protocol, and the absorbance of each sample was measured at a wavelength of 530 nm.

Statistical analysis

All statistical analyses were performed with SPSS 22.0 (IBM, USA). All results are expressed as the mean ± SD. The difference between the means of more than two groups was analyzed by one-way analysis of variance (ANOVA). When ANOVA showed significant differences, pairwise comparisons of means were made by Bonferroni’s post hoc t-test with correction. A value of P < 0.05 was considered statistically significant.

Establishment of a TDP-43-transfected SH-SY5Y cell model

Cell were transfected with virus an MOI of 0, 10, 20, 30, 40, 50 or 60. Cells transfected at an MOI of 0 were in good condition and showed no green fluorescence under a fluorescence microscope. Cells transfected at an MOI of 30 were in good condition, showed clearly visible dendrites, fluoresced under a fluorescence microscope, and showed high transfection efficiency. The fluorescence and transfection efficiency of cells transfected at MOIs of 10 and 20 were lower than those transfected at an MOI of 30. The cells transfected at MOIs of 40 and 50 were in a poor state; there cell bodies were crumpled and collapsed, and some of the cells were dead. Cells transfected at an MOI of 60 were blurred or absent, and a large number of cells were dead. In conclusion, the optimal MOI was determined to be 30 (Fig. 1A). The morphology of cell transfected with TDP-43 for 12, 24 and 48 h were observed, and the cells transfected for 12 h showed the most normal morphology, although some shrunken cells were observed. At 24 h, the cells were in a poor state; most of the cells were crumpled or collapsed, and some were dead. At 48 h, the cells were in a worse state, and a large number of cells were dead (Fig. 1B). After transfection for 8, 10, 12, 14 or 16 h, the cell activity of the control group and TDP-43 group was assessed, and the results showed that after transfection for 12, 14 and 16 h, the activity of the TDP-43 group was decreased compared with that of the Con group, indicating that cell activity was reduced after transfection with TDP-43 and that the damage model was successfully established. TDP-43 had strong cytotoxicity, and the cells were in a poor state at 24 h and 48 h after transfection. However, at 12 h after transfection, the cells were in a relatively healthy state, although some cells showed morphological changes and cell activity was significantly decreased. Therefore, this time point was suitable for assessing the effect of ICT. In this experiment, cells were transfected with TDP-for 12 h (Fig. 1C). The mRNA expression level of TDP-43 in the TDP-43 group was higher than that in the control group (Fig. 1D). Western blotting was used to measure the expression of transfected TDP-43 protein, and the results showed that TDP-43 protein expression in the TDP-43 group was increased compared with that in the control group (Fig. 1E). In summary, an MOI of 30 and a transfection time of 12 h were selected to establish the TDP-43-transfected cell model.

Effects of ICT at different concentrations for different durations on the activity of TDP-43-transfected SH-SY5Y cells

After modeling, ICT at concentrations of 0.0, 0.1, 1.0 and 10.0 µmol/L was added, and the cell activity of each group at 12, 24 and 48 h were determined. The results showed that optimal cell viability was obtained at a drug concentration of 1.0 µmol/L and a treatment time of 48 h. Therefore, subsequent experiments were carried out under these conditions (Fig. 2A).

Effects of ICT on the viability of SH-SY5Y cells transfected with TDP-43

Changes in cell activity in the control group, control + ICT group, TDP-43 group, and TDP-43 + ICT group were assessed after treatment with 1.0 µmol/L ICT for 48 h. The results showed that compared with that of the control group, the activity of the TDP-43 group was decreased, while the cell activity of the TDP-43 + ICT group was increased after ICT treatment (Fig. 2B).

Effects of ICT on TDP-43 expression in SH-SY5Y cells transfected with TDP-43

The Western blot results showed that compared with that in the Con group, the expression level of TDP-43 in the TDP-43 group was increased; however, after ICT treatment, the expression level of TDP-43 decreased (Fig. 3).

Effects of ICT on ATP content, the MMP and CytC expression in SH-SY5Y cells transfected with TDP-43

JC-1, which can quickly and sensitively detect MMP changes, was used a fluorescent probe in this experiment. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form a polymer that emits strong red fluorescence. In unhealthy mitochondria, due to a decrease in the MMP, JC-1 can only exist in the cytoplasm as monomers, which produce green fluorescence. The results showed that compared with the control group and control + ICT group, the TDP-43 group showed reduced red fluorescence, enhanced green fluorescence and a decreased MMP. Green fluorescence was weakened, red fluorescence was enhanced, and the MMP was increased in the TDP-43 + ICT group compared with the TDP-43 group (Figs. 4A and 4B).

The results showed that there was no significant difference in ATP content between the control + ICT group and control group and that ATP content in the TDP-43 group was lower than that in the control group. Compared with that in the TDP-43 group, the ATP content in the TDP-43 + ICT group was increased (Fig. 4C).

The Western blot results showed that compared with that in the control group, the expression level of CytC in the TDP-43 group was increased, while the expression level of CytC decreased after ICT treatment (Fig. 4D).

The effects of ICT on T-AOC, GSH-Px activity, T-SOD activity, CuZn-SOD activity, Mn-SOD activity and MDA content in TDP-43-transfected SH-SY5Y cells

T-AOC (Fig. 5A), GSH-Px activity (Fig. 5B), T-SOD activity (Fig. 5C), CuZn-SOD activity (Fig. 5D) and Mn-SOD activity (Fig. 5E) were reduced in SH-SY5Y cells transfected with TDP-43 compared with control cells. After ICT treatment, T-AOC (Fig. 5A), GSH-Px activity (Fig. 5B), T-SOD activity (Fig. 5C), CuZn-SOD activity (Fig. 5D) and Mn-SOD activity (Fig. 5E) were increased. MDA content was increased in TDP-43-transfected cells compared with control cells (Fig. 5F) and decreased after ICT treatment (Fig. 5F).