Sch B was bought from Shanghai Yuanye Biotechnology Co., Ltd., and TMZ from Shanghai Maclin Biochemical Technology Co., Ltd. DMEM medium and penicillin/streptomycin mixture were acquired from Sigma (St. Louis, MO, USA). Fetal bovine serum was purchased from Thermo Fisher Technologies (Waltham, MA, USA); The caspase inhibitor Z-VAD-FMK was obtained from MCE (Monmouth Junction, NJ, USA); and Kits for malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) assays were obtained from Jianjieng Bioengineering Institute of Nanjing (Nanjing, China). The kit for detecting reactive oxygen species (ROS) was bought from Shenyang Wanxiang Biological Co., Ltd., while the BCA protein concentration kit, Hoechst 33258 dye solution, Mitochondrial Permeability Conversion Pore (MPTP) assay kit, Annexin V-FITC PI Apoptosis Kit, mitochondrial membrane Potential (MMP) assay Kit, and cell cycle assay kit, were all purchased from Shanghai Biyuntian Biotechnology Company. Equipment included a 5% CO₂ incubator (ThermoFisher Technology Co., Ltd., Waltham, MA, USA), a fluorescence microscope (Leica TCS SP8, Wetzlar, Germany), a flow cytometer (FACSCalibur, San Jose, CA, USA), a gel imaging system (Tanon 5200, Shanghai Tienang Technology Co., Ltd., Shanghai, China), and a full-wavelength enzyme microscope (Epoch, BioTek, Winooski, VT, USA).

Human astroblastoma U87 cells (ATCC® HTB-14™) were acquired from the American Type Culture Collection and their identity was verified by short tandem repeat (STR) profiling. The cells were maintained in a humidified incubator at 37 °C with 5% CO₂, cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Routine mycoplasma testing was performed using the MycoAlert™ Detection Kit (Walkersville, MD, USA), with all tests returning negative. For experimental use, cells were harvested upon reaching the logarithmic growth phase and the resulting cell suspension was seeded into either 6-well or 96-well plates.

The concentrations of MDA, SOD, CAT, and GSH in the cell homogenate supernatant were determined with respective assay kits following the kit protocols. ROS levels in U87 cells were assessed using a DCFH-DA (2^′, 7^′-dichlorofluoroscin diacetate) probe. After adding 5 µL of DCFH-DA (1 µM) fluorescent dye to each well, the cells were incubated in the dark in 30 min at 37 °C, then rinsed twice with phosphate-buffered saline (PBS). ROS generation was detected by fluorescence microscopy (Leica TCS SP8, Wetzlar, Germany).

Changes in MMP in U87 cells were assessed with the JC-1 assay kit following the manufacturer’s protocol. Briefly, cells seeded in 6-well plates were subjected to drug treatments for 24 h. After being rinsed with PBS, the cells were incubated with the working JC-1 staining solution at 37 °C for 30 minutes. Subsequently, unbound dye was removed by washing with the provided 1$\times$ assay buffer. The fluorescence intensity was then visualized and captured using a Leica TCS SP8 fluorescence microscope to determine the MMP alterations.

To assess nuclear morphological changes in U87 cells, the Hoechst 33258 staining kit was employed. After fixation with 4% paraformaldehyde, cells were subjected to PBS washes and permeabilized in 0.2% Triton X-100. Nuclear staining was subsequently performed by incubating with Hoechst 33258 (10 µg/mL) for 5 min following an additional PBS wash. The stained cells were then observed under a fluorescence microscope (Leica TCS SP8), with bright blue fluorescence indicating the cell nuclei. Quantitative analysis of the images was performed using Image pro plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

The Annexin V-FITC/PI double-staining method was utilized to detect apoptosis. Cells were harvested, washed twice with PBS, and the resulting cell pellet was collected by centrifugation. The culture medium was discarded, and cells were digested with trypsin. After digestion, cells were transferred to a centrifuge tube, washed with PBS, and centrifuged again to obtain a cell pellet. The pellet was then resuspended in buffer, and FITC Annexin V staining solution (green fluorescence) was added. The admixture was gently mixed and incubated in the dark for 5 minutes. Subsequently, the PI staining solution (red fluorescence) was added and incubated for 5 minutes. After staining, PBS was added in a volume appropriate to the cell count, and the proportion of apoptotic cells was analyzed using flow cytometry (FACSCalibur, San Jose, CA, USA).

The culture medium was aspirated from the 6-well plate. Cultures were supplemented with Calcein AM and the corresponding fluorescence quenching reagent at specified volumes, following the MPTP detection kit guidelines. Following a 45 min incubation at 37 °C in the dark, the staining solution was exchanged for fresh pre-warmed culture medium and the cells were incubated for an additional 30 min under identical conditions. Removed the medium and rinsed the wells 2–3 times with PBS. Detection buffer was then added before observation under a fluorescence microscope.

After centrifugation and PBS resuspension, cells were subsequently fixed through incubation in 1 mL of pre-cooled 70% ethanol for 2 h at 4 °C, as directed by the Cell Cycle and Apoptosis Detection Kit. Following fixation, the cells were centrifuged, and the pellet was collected. Each sample was then resuspended in 0.5 mL of propidium iodide (PI) staining solution and incubated in a water bath at 37 °C for 30 min in the dark. Samples were analyzed by flow cytometry using a 488-nm excitation wavelength.

Cells were allocated into four experimental groups: control, TMZ treated, Sch B treated, and TMZ + Sch B co-treated. Each group was allocated five wells. After treatment, cells were plated in 96-well plates at 6 $\times$ 10⁴ cells/well. After adding 10 µL MTT solution per well, the plates were incubated for 2 h. The absorbance of each well was subsequently determined at 450 nm using a microplate reader.

U87 cells were lysed for 2 h, and the protein concentration was measured using a BCA assay kit. The protein lysate was boiled at 100 °C for 7 min for denaturation. The samples were resolved by SDS-PAGE and subsequently transferred to a PVDF membrane. The membrane was then blocked with 5% non-fat milk for 2 h. Primary antibodies were incubated with the membrane at 4 °C overnight to detect proteins of interest: Cytochrome c (1:1000, 66264-1-IG, Proteintech, Wuhan, China), Bax (1:4000, 50599-2-ig, Proteintech), (cleaved)-Caspase E9 (1:1000, 66169-1-ig, Proteintech), Bcl-xL (1:100,000, 10773-1-AP, Proteintech), Bcl-2 (1:4000, 68103-1-ig, Proteintech), cleaved-caspase3 (1:10,000, 82202-1-RR, Proteintech), CDK4 (1:4000, 11026-1AP, Proteintech), P21 (1:2000, 10355-1-AP, Proteintech), P53 (1:10,000, 60283-2-IG, Proteintech), and GAPDH (1:50,000, 60004-1 1-Ig, Proteintech). Following the incubation period, the membrane was rinsed with TBST to remove unbound antibodies. Subsequently, it was probed with the corresponding HRP-conjugated secondary antibody for 1 h. The protein bands were colored using Emitter Coupled Logic (ECL) and analyzed by Image pro plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

Statistical analyses were conducted with GraphPad Prism 8.0.4 (GraphPad Software, San Diego, CA, USA). All experiments included three independent biological replicates (n = 3) with triple technical replicates per condition. Data are presented as mean $\pm$ standard deviation (SD). Group comparisons were performed using unpaired two-tailed Student’s t-test (two groups) or one-way ANOVA with Tukey’s post-hoc test (multiple groups). Significance levels are indicated as *p 0.05, **p 0.01, and ***p 0.001.

The effects of TMZ, Sch B, and their combination on U87 cell proliferation were analyzed using an MTT assay. Varying doses of TMZ, Sch B and TMZ + Sch B were used to treat cells for 48 h followed by MTT incubation to evaluate cell viability. TMZ (0–400 µM) exerted a dose-dependent inhibitory effect on U87 cell proliferation (Fig. 1A). At a concentration of 200 µM, TMZ reduced U87 cell viability to 72.47% compared to the control group. Similarly, Sch B suppressed U87 cell proliferation over a concentration range of 0–1600 µg/mL (Fig. 1B), reducing cell viability to 70.4% at 200 µg/mL. When a constant concentration of TMZ (200 µM) was combined with increasing concentrations Sch B, U87 cell viability further de-creased (Fig. 1C). Notably, the combination of 200 µM TMZ and 200 µg/mL Sch B reduced U87 cell viability to 54.14%, producing a more pronounced inhibitory effect than either agent alone, which suggests a synergistic effect between Sch B and TMZ.

Fig. 1.

Cell viability was assessed by MTT assay. (A) Effects of TMZ on U87 cell viability. (B) Effects of Sch B on U87 cell viability. (C) Effects of TMZ and Sch B combination on U87 cell viability. Data are presented as mean $\pm$ SD. Statistical significance is denoted as follows: *p 0.05, **p 0.01, ***p 0.0001. TMZ, Temozolomide; Sch B, Schisandrin B.

Hoechst staining showed that the integration of TMZ and Sch B resulted in significantly brighter blue fluorescence compared to either treatment alone, indicating enhanced apoptosis in U87 cells (Fig. 2A). Flow cytometry further confirmed the enhanced apoptosis induced by the combined treatment (Fig. 2B). Western blot indicated that the combination treatment modulated the expression of key apoptotic regulators relative to single-agent treatments. Specifically, it elevated pro-apoptotic proteins (Bax, cytochrome c) while reducing anti-apoptotic proteins (Bcl-2, Bcl-xL). This shift in the balance was accompanied by activation of caspase-3 and caspase-9, thereby promoting apoptosis in U87 cells (Fig. 2C). Additionally, JC-1 staining showed a reduction in the red/green fluorescence ratio, suggesting a decrement in the MMP. While both TMZ and Sch B individually caused a decline in mitochondrial membrane potential, their combination led to a more pronounced effect, further accelerating apoptosis in U87 cells (Fig. 2D). These results suggest that TMZ and Sch B act synergistically to enhance apoptotic signaling in glioma cells, likely through amplification of the intrinsic mitochondrial pathway. This combination may provide a promising therapeutic approach for glioblastoma by improving treatment efficacy and potentially overcoming resistance to standard chemotherapy.

Fig. 2.

Effect of TMZ and Sch B combination on U87 cell apoptosis. (A) Hoechst 33342 staining shows condensed and fragmented nuclei indicating apoptosis. Scale bar = 100 µm. (B) Flow cytometric analysis quantifies apoptotic cells. (C) Analysis of apoptosis-related protein expression by Western blot. (D) Detection of mitochondrial membrane potential changes by JC-1 staining. Scale bar = 100 µm. Data are presented as mean $\pm$ SD (n = 3). Statistical significance is reported at *p 0.05, **p 0.01, and ***p 0.001.

Mitochondrion acts as a key regulator of the intrinsic apoptotic pathway. The maintenance of mitochondrial integrity is largely influenced by the MPTP, a non-selective channel connecting the inner and outer mitochondrial membranes, whose opening permits the release of pro-apoptotic factors that drive cell death. In this study, MPTP staining revealed that treatment with either TMZ or Sch B resulted in moderate pore opening, as indicated by de-creased green fluorescence. However, the combination treatment of TMZ and Sch B led to a more pronounced loss of fluorescence, suggesting a significant increase in opening of the MPTP (Fig. 3A). Sustained MPTP opening is known to trigger mitochondrial oxidative stress by increasing the level of reactive oxygen species (ROS) accumulation. Staining for ROS revealed a marked rise in level of intra-cellular ROS following the pretreatment of TMZ and Sch B, as evidenced by enhanced green fluorescence (Fig. 3B). This result indicates an aggravation of oxidative stress. Notably, the combination group exhibited a marked increase in MDA levels, an indicator of lipid peroxidation, relative to the individual treatments, suggesting severe mitochondrial membrane impairment (Fig. 3C).

Fig. 3.

Effect of TMZ/Sch B combination on the mitochondrial function of U87 cells. (A) MPTP staining evaluates mitochondrial permeability transition pore opening. Scale bar = 100 µm. (B) ROS levels measured by DCFH-DA staining. Scale bar = 100 µm. (C) Lipid peroxidation assessed via MDA content. (D–F) Antioxidant enzyme activities of SOD, CAT, and GSH, respectively. Data are presented as mean $\pm$ SD. *p 0.05, **p 0.01, and ***p 0.001. MPTP, Mitochondrial Permeability Conversion Pore; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GSH, glutathione.

To further evaluate the redox status, the levels of key intracellular antioxidants such as SOD, CAT, and reduced GSH were measured. A substantial decline in these antioxidant defenses was observed in the combination treatment group, indicating compromised antioxidant capacity and a shift toward a pro-oxidant state (Fig. 3D–F). Together, our data indicate that the TMZ/Sch B enhances mitochondrial dysfunction through increased MPTP opening, elevated ROS production, and impaired antioxidant defense, thereby amplifying oxidative stress-mediated apoptosis in glioma U87 cells.

Cyclin-dependent kinase 4 (CDK4) is important for regulating the G1 to S phase transition during cell-cycle progression. In response to DNA impairment, the tumor suppressor protein p53 promotes transcription of the CDK inhibitor p21, which subsequently inhibits CDK activity and halts progression of cell cycle. Western blot analysis revealed that combined treatment with TMZ and Sch B significantly reduced CDK4 expression and upregulated p53 and p21 levels when compared with controls, indicating disruption of normal cell cycle regulation (Fig. 4A) (p 0.05). Flow cytometric detection showed that TMZ + Sch B caused notable arrest of U87 cells at the G2/M transition, indicating effective cell cycle blockade at this phase (Fig. 4B).

Fig. 4.

Effect of TMZ/Sch B on the U87 cell cycle. (A) Western blot analysis of CDK4, p53, and p21 protein expression. (B) Flow cytometry of DNA content showing changes in cell cycle phases. Quantification based on triplicate experiments, *p 0.05, **p 0.01, and ***p 0.001.

Z-VAD-FMK, a broad-spectrum and cell-permeable caspase inhibitor, is widely used to block caspase-dependent apoptosis. To verify whether TMZ + Sch B-induced cell death in U87 cells occurs through a caspase-dependent pathway, cells were pretreated with Z-VAD-FMK before combination treatment. Hoechst 33342 staining (Fig. 5A) confirmed the inhibitory effect of Z-VAD-FMK on apoptosis, showing fewer condensed nuclei and reduced bright-blue fluorescence in the TMZ + Sch B + Z-VAD-FMK group compared with the TMZ + Sch B group. Flow-cytometric analysis (Fig. 5B) supported these findings, showing that the apoptotic rate increased to 19.3% following TMZ + Sch B treatment but decreased to 13.8% after Z-VAD-FMK co-treatment, confirming that the inhibitor effectively reduced combination-induced apoptosis. Western blot analysis (Fig. 5C) showed that Z-VAD-FMK increased anti-apoptotic protein Bcl-2 while reducing pro-apoptotic markers (Bax, cytochrome c, cleaved caspase-9, and cleaved caspase-3), indicating suppression of caspase activation. JC-1 staining (Fig. 5D) demonstrated that TMZ + Sch B treatment caused a pronounced loss of mitochondrial membrane potential (MMP), whereas co-treatment with Z-VAD-FMK partially restored MMP, as indicated by an elevated red/green fluorescence ratio. Collectively, these results demonstrate that TMZ + Sch B induces caspase-dependent apoptosis in U87 cells, which can be partially reversed by Z-VAD-FMK.

Fig. 5.

Effects of Z-VAD-FMK on apoptosis in U87 cells induced by TMZ and Sch B. (A) Hoechst staining reveals nuclear changes. Scale bar = 100 µm. (B) Flow cytometry quantifies apoptotic rate. (C) Western blot showing caspase activation. (D) JC-1 staining visualizes mitochondrial potential restoration by Z-VAD-FMK. Scale bar = 100 µm. Data shown as mean $\pm$ SD (n = 3). *p 0.05, **p 0.01, and ***p 0.001.

To assess the role of caspase inhibition in mitigating mitochondrial dysfunction, U87 cells were co-treated with Z-VAD-FMK alongside TMZ and Sch B. MPTP staining demonstrated increased green fluorescence in the presence of Z-VAD-FMK compared to the TMZ + Sch B group alone, indicating reduced MPTP opening and improved mitochondrial integrity (Fig. 6A). Similarly, ROS staining revealed diminished green fluorescence following Z-VAD-FMK treatment, indicating a significant reduction of intracellular ROS and alleviation of oxidative stress (Fig. 6B). Lipid peroxidation, assessed via MDA levels, was significantly lower in the Z-VAD-FMK-treated group compared to cells treated with TMZ + Sch B alone, indicating reduced mitochondrial membrane damage (Fig. 6C). Moreover, the levels of key intracellular antioxidants (SOD, CAT, and GSH) were significantly elevated in the Z-VAD-FMK co-treatment group, reflecting enhanced antioxidant defense and restoration of mitochondrial function (Fig. 6D–F).

Fig. 6.

Effects of Z-VAD-FMK on mitochondrial function in U87 cells treated with TMZ and Sch B. (A) MPTP. Scale bar = 100 µm. (B) ROS. Scale bar = 100 µm. (C) MDA. (D) SOD. (E) CAT. (F) GSH levels were measured to assess mitochondrial damage and oxidative stress. Z-VAD-FMK reduced oxidative markers and restored antioxidant enzyme levels. *p 0.05, **p 0.01, and ***p 0.001 indicates statistical significance.

This study was conducted exclusively using the U87 glioma cell line. Although well-characterized, these cells may not fully represent the molecular and phenotypic diversity of glioblastoma. Consequently, the results should be interpreted as preliminary evidence of synergistic and pro-apoptotic effects in vitro, rather than as confirmation of resistance reversal. In addition, quantitative assessment of the synergistic interaction (e.g., using a Chou–Talalay synergy index) was not performed and should be included in future studies to substantiate the combined effect. Future investigations that incorporate additional glioma cell lines (e.g., T98G, U251, and LN229) and patient-derived primary cultures will be essential to validate these findings across diverse tumor backgrounds. Moreover, in-vivo studies with xenograft or orthotopic glioma models are needed to evaluate pharmacokinetic interactions, tumor microenvironment effects, and systemic safety of the Sch B + TMZ combination. Despite these limitations, the current work provides a mechanistic foundation that supports the potential of Sch B as an adjunct agent to enhance TMZ efficacy in glioma therapy.