MPP⁺ was obtained from Macklin, China; 2-ABP was obtained from APExBIO, United States; reference substance of Rutin was obtained from Yuanye, China; reference substance of echinacoside was obtained from Alfa, China; reference substance of paeoniflorin, salvianolic acid B, acteoside and tanshinone IIa were obtained from Dilger, China; CCK-8, Fluo-3-AM fluorescent probe, Reactive oxygen species (ROS) assay kit, and JC-1 mitochondrial membrane potential (MMP) assay kit were obtained from Beyotime, China; Rhod-2-AM cell permeable calcium ion fluorescence probe Yeasen, China; IP₃R, VDAC, GRP75, Cyt-C, Bax, Bcl-2, Caspase-3, and Caspase-9 antibodies were obtained from Proteintech, China; Cell apoptosis kit and MCU antibodies were obtained from Abbkine, China; MCU antibodies and BCA Protein Assay Kit were obtained from Boster, China; DMEM medium was obtained from Gibco, United States; Origin fetal bovine serum (FBS) was obtained from MRC, United States.

The study has been approved by the ethics committee of Fujian University of Traditional Chinese Medicine (No. 2020063), and all operations were carried out in adherence to the Guide for the Care and Use of Laboratory Animals. Forty Sprague-Dawley rats (SD rats), SPF grade, were obtained from Slac Laboratory (certificate 20170005022867). Rats were raised in an SPF-grade animal laboratory with a relative humidity of 60%, 25°C, and a 12-h light/dark cycle.

CRSJG is a new traditional Chinese medicine developed by the Creation of Major New Drugs for Major National Science and Technology Projects (No.2019ZX09301154), and it was obtained from the Third Affiliated People’s Hospital of Fujian University of Traditional Chinese Medicine. The compound ingredients include Cistanche deserticola Y.C. Ma (Rou congrong, Orobanchaceae), Polygonatum kingianum Collett & Hemsl. (Jiu huangjing, Asparagaceae), Salvia miltiorrhiza Bunge (Dan shen, Lamiaceae), Paeonia lactiflora Pall. (Chi shao, Paeoniaceae), and Paeonia suffruticosa Andrews (Mu danpi, Paeoniaceae), the dosage ratio of each component is 6:12:15:12:10, and related details are summarized in Table 1 (Ou et al., 2022).

The total amount of the five materials was 275 g, including Cistanche deserticola (30 g), Polygonatum kingianum (60 g), Salvia miltiorrhiza (75 g), Paeonia lactiflora (60 g), and Paeonia suffruticosa (50 g). We added 2,750 mL and 2,200 mL of water sequentially, and decocted the mixture twice at 100°C for 1 h each time. After filtering the residue, 5% dextrin was added, mixed thoroughly, and dried to yield CRSJG.

After 1 week of adaptive feeding, rats were randomly divided into drug-containing serum group (n = 14) and blank serum group (n = 14). The CRSJG-medicated serum group received CRSJG via oral gavage at a dose of 10 g/(kg·d) for seven consecutive days, while the blank serum group received an equal volume of normal saline. Following the administration period, all rats were fasted for 12 h before anesthesia but were provided unrestricted access to water. All rats were injected with a dose of urethane (1,000 mg/kg), and blood was collected from the abdominal aorta. Rat-serum was collected, inactivated, and stored at −80°C.

Rats were randomly selected from the drug-containing serum group (n = 6) and the blank serum group (n = 6). These rats were fasted for 12 h but allowed free access to water. They received 10 g/(kg·d) of CRSJG or an equivalent dose of normal saline via intragastric administration. Orbital blood samples were collected at 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24, and 36 h post-administration. Reference substances were weighed and dissolved in methanol by ultrasonication to prepare the reference solutions. The concentrations of these solutions were: echinacoside 126,819 ng/mL, paeoniflorin 2,657 ng/mL, salvianolic acid B 2,563 ng/mL, acteoside 603.5 ng/mL, tanshinone IIa 137.1 ng/mL, and rutin 457.5 ng/mL.

Chromatographic analysis: Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μmol/L). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid in water (B), with a gradient elution at a flow rate of 0.3 mL/min. The column temperature was maintained at 40 °C, and the injection volume was 2 μL. The gradient profile was as follows: from 0.0 to 1.5 min, 14%–30% A; from 1.5 to 3.0 min, 30%–70% A; from 3.0 to 5.0 min, 70%–90% A; from 5.0 to 6.0 min, held at 90% A; from 6.0 to 6.1 min, 90%–14% A; and from 6.1 to 7.0 min, held at 14% A.

Mass spectrometry: electrospray ionization source (ESI); Capillary ionization voltage 3.5 kV; Ion source temperature 110°C; Desolvent gas N₂, volume flow rate 800 L/h, 500°C; Back-blowing gas N₂, volume flow 50 L/h; Collision gas Ar, volume flow 0.13 mL/min; Mass spectrum data acquisition and processing by Masslynx4.1 software, multiple reaction monitoring (MRM) scanning, and related mass spectrum conditions are shown in Table 2.

Calibration standards were prepared by diluting methanol stock to yield a series of gradient reference solutions. 200 μL of each standard was dried under a gentle nitrogen stream at 45°C, then reconstituted in 200 µL of the blank serum. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze these samples, construct standard curves, and assess linearity. To validate the method, three quality-control levels (low, medium, high) of internal-standard mixtures were spiked into blank serum, and the following performance characteristics were evaluated: precision tests, recovery assays, extraction recovery assays, matrix effect analyses, and sample stability. Pharmacokinetic parameters were determined in CRSJG-medicated serum collected at defined intervals post-intragastric administration, and were calculated using DAS 2.0 software with a non-compartmental model.

Human neuroblastoma cell line (SH-SY5Y) was obtained from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were cultured in DMEM high-glucose medium (10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) at saturated humidity, 5% CO₂, and 37°C.

Cell Counting Kit-8 (CCK-8) Assay was used to screen the induction concentration of MPP⁺. MPP⁺ (0, 250, 500, 750, 1,000, 1,500, 2000 μmol/L) was added to the cells, and a blank control group (without cells or drugs) was included to account for background absorbance. After 24 h of intervention, 10 μL CCK-8 (Beyotime, China) was added, incubated for 2 h, and the optical density (OD) at 450 nm was measured using a microplate reader (Bio-TEKEL, United States).

For CRSJG-medicated serum intervention, SH-SY5Y cells were pre-treated with 1,000 μmol/L MPP⁺ for 24 h, followed by exposure to 0%, 2.5%, 5%, 10%, 15%, or 20% CRSJG-medicated serum. Three zero adjustment holes (without cells or drugs) were included. At 12 h, 24 h, 36 h, and 48 h post-treatment, 10 μL CCK-8 solution was added to each well, followed by 2 h of incubation under the same conditions. The OD at 450 nm was recorded using a microplate reader. OD values of the Background absorbance controls (wells containing only the medium and CCK-8 reagent) were subtracted from experimental readings to ensure data accuracy.

SH-SY5Y cells were divided into MPP⁺ group, 2-aminoethoxydiphenyl borate (2-APB) group, CRSJG group with 1,000 μmol/L MPP⁺, control group without MPP⁺. 2-APB is a classic cell-penetrable inhibitor of IP₃R that can block IP₃-mediated intracellular calcium release, and was used as a positive control drug in our study.

SH-SY5Y cells were seeded at a density of 1 × 10⁵ cells/mL in culture dishes, and intervention begins when cells density reached 70–80 percent. CRSJG-medicated serum was added to CRSJG group, and blank serum was added to control group, MPP⁺ group, and 2-APB group. We ensured that the total serum concentration (CRSJG-medicated serum or blank serum + FBS) at 10% in all groups. The cells were cultured for 24 h with 5% CO₂, 37°C. The intervention system of each group was as follows: Control group (7.5% FBS +2.5% blank serum); MPP⁺ group (7.5% FBS +1000 μmol/L MPP⁺ + 2.5% blank serum); 2-APB group (7.5% FBS +1000 μmol/L MPP⁺ + 2.5% blank serum +100 μmol/L 2-APB); CRSJG group (7.5% FBS +1000 μmol/L MPP⁺ + 2.5% CRSJG-medicated serum). MPP⁺ and 2-APB were dissolved directly in high-glucose DMEM to achieve the required working concentrations.

The intracellular Ca²⁺ levels were detected by fluorescence probe and flow cytometry. After induction with MPP⁺ and subsequent treatment with either CRSJG-medicated serum or blank serum, SH-SY5Y cells were incubated with 300 μL of the mitochondrial Ca²⁺-sensitive fluorescent dye Rhod-2 AM (Yeasen, China; experimental concentration 10 μmol/L) and 300 μL of the cytoplasmic Ca²⁺-sensitive fluorescent dye Fluo-3 AM (Beyotime, China; experimental concentration 10 μmol/L) for 30 min at 37°C in the dark. Following incubation, cells were washed twice with PBS, and fluorescence images were acquired using a fluorescence microscope (Nikon, Japan). The mean fluorescence intensity (MFI) was quantified using ImageJ software.

For flow cytometry analysis of intracellular Ca²⁺ Levels, SH-SY5Y cells stained using the same protocol were gently dissociated into a single-cell suspension using a pipette, centrifuged at 300 g for 3 min, and then resuspended in 200 μL of PBS. The average fluorescence intensity of FITC (for Fluo-3 AM) or PE (for Rhod-2 AM) channels was recorded by flow cytometry (Beckman Coulter, United States).

Intracellular ROS levels in SH-SY5Y cells were measured using ROS assay kit (Beyotime, China), following the manufacturer’s instructions. SH-SY5Y cells were collected and prepared as a single-cell suspension. For the positive control group, 1 μL of Rosup was added and incubated for 20 min to induce ROS production. The negative control group received no treatment. All other experimental groups followed the previously described intervention protocol. After drug treatment, cells were incubated with 10 μmol/L DCFH-DA diluted in PBS at 37°C for 30 min in the dark. Following incubation, cells were washed three times with PBS to remove excess dye. Intracellular ROS levels were then quantified using flow cytometry (FITC). Detailed gating strategies are shown in Annex 1.

MMP was evaluated using a JC-1 assay kit (Beyotime, China) in accordance with the manufacturer’s protocol. SH-SY5Y cells were collected and prepared as a single-cell suspension. The positive control group was treated with 10 μmol/L CCCP for 20 min. The negative control group remained untreated. Other groups received the same interventions as described previously. Following treatment, cells were incubated with JC-1 working solution (1×) at 37°C for 30 min in the dark. Cells were then washed twice with JC-1 buffer and analyzed by flow cytometry. JC-1 aggregates (high MMP) were detected in the PE channel, while JC-1 monomers (low MMP) were measured in the FITC channel. Detailed gating strategies are shown in Annex 1.

Cell apoptosis was assessed using an Annexin V-FITC/PI Apoptosis Detection Kit (Abbkine, China). After treatment, SH-SY5Y cells were harvested, washed twice with cold PBS, and resuspended in 500 μL of binding buffer. Cells were then stained with 5 μL Annexin V-FITC and 10 μL PI and incubated at 37°C for 30 min in the dark. Single-staining controls were prepared using Annexin V-FITC or PI only, and untreated cells served as the negative control. Samples were analyzed by flow cytometry, and the percentages of early and late apoptotic cells were quantified. Detailed gating strategies are shown in Annex 1.

Western blotting was performed to detect proteins related to apoptosis and Ca²⁺ transport. SH-SY5Y cells were trypsinized with 0.25% trypsin-EDTA, collected by centrifugation at 1,200 rpm for 5 min at 4°C, and washed twice with ice-cold PBS. The cell pellet was resuspended in RIPA lysis buffer (containing 1% PMSF and protease/phosphatase inhibitors), followed by incubation on ice for 15 min with intermittent vortexing. Lysates were then centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant containing total protein was collected.

Protein concentration was determined using the BCA Protein Assay Kit (Boster, China). Equal amounts of protein were mixed with 1× SDS loading buffer and denatured at 95°C for 10 min. Samples were separated on a 10% SDS-PAGE gel and transferred onto 0.45 μm PVDF membranes (Millipore, United States). Membranes were blocked with 5% non-fat milk in PBS for 2 h at room temperature, followed by incubation with primary antibodies (IP3R, VDAC, GRP75, Cyt-C, Bax, Bcl-2, Caspase-3, and Caspase-9 antibodies were obtained from Proteintech, China); MCU antibodies were obtained from Abbkine, China; Dilution Ratio 1:1,000) overnight at 4°C with gentle shaking.

After washing, membranes were incubated with HRP-conjugated secondary antibodies (Proteintech, China; Dilution Ratio 1:10,000) for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) detection system and analyzed using ImageLab software (Bio-rad, United States).

SH-SY5Y cells cultured in each group were fixed with 2.5% glutaraldehyde for 5 min. Cells were scraped off with the cell scraper, centrifuged for 15 min, 2000 rpm, and then covered with electron microscopy fixing solution, so that the cells were completely suspended in the fixing solution, and fixed for 30 min. After dehydrating with acetone, samples were embedded and sectioned into ultrathin slices, which were stained with lead citrate for 10 min and uranium acetate for 30 min. After washing and drying, the stained cells samples were scanned and observed by transmission electron microscope (Hitach, Japen).

All experimental data were processed and analyzed by SPSS 24.0, and presented as mean ± standard deviation. The Kolmogorov-Smirnov (K-S) test was used to assess the normality of data distribution. If the data conformed to a normal distribution, One-way analysis of variance (ANOVA) was performed to compare differences among groups; the Least Significant Difference (LSD) method was applied when variances were homogeneous; the Games-Howell method was used when variances were heterogeneous. If the data did not follow a normal distribution, the Kruskal-Wallis test (non-parametric independent sample rank-sum test) was used instead. The test level was α = 0.05, and p < 0.05. Finally, GraphPad Prism version 8.0.2 (263) was used to complete each experimental statistical chart.

Methodological analysis demonstrated that the chromatographic peaks of the primary constituents and rutin were distinctly separated, with no interference from endogenous substances (Figures 2A–C). The recovery and extraction assays for each main component and rutin met established standards. Additionally, rat serum exhibited no significant interference with the primary components of CRSJG and rutin (Figure 2D). These findings comply with the criteria for biological sample analysis. Pharmacokinetic profiling indicated the presence of CRSJG’s key markers - echinacoside, paeoniflorin, salvianolic acid B, acteoside, and tanshinone IIa,- in CRSJG-medicated serum. The mean quantitative results showed that the plasma concentration of the drug peaked at 0.25 h (15 min) post-administration: echinacoside at 718.7 ng/mL, paeoniflorin at 462.0 ng/mL, salvianolic acid B at 373.5 ng/mL, acteoside at 10.61 ng/mL, and tanshinone IIa at 75.24 ng/mL (Figure 2E). The detailed results of the pharmacokinetic parameters of CRSJG-medicated serum are listed in the supplementary figure.

We investigated the impact of MPP⁺ on cell proliferation independently. The findings indicated that all tested concentrations of MPP⁺ significantly decreased cell proliferation, with an EC₅₀ value of 1,110 μmol/L (Figure 3A). Therefore, 1,000 μmol/L MPP⁺, being closest to the EC₅₀ value, was selected for subsequent experiments. Subsequently, we applied varying concentrations of CRSJG-medicated serum to MPP⁺ induced cells over different durations. Compared to the blank control group, which received blank serum intervention, any concentration of CRSJG-medicated serum was found to effectively mitigate the MPP⁺ induced reduction in cell proliferation rate after an intervention exceeding 12 h. Notably, a 2.5% CRSJG-medicated serum demonstrated the most significant enhancement in proliferation at 24 h of intervention, thereby establishing the most optimal treatment condition (Figures 3B,C). This criterion was therefore chosen for application in further experiments.

We respectively employed Rhod-2 and Fluo-3 fluorescence probes to measure mitochondrial-Ca²⁺ (mito-Ca²⁺) and cytoplasmic-Ca²⁺ (cyto-Ca²⁺) levels respectively, followed by a quantitative analysis of mean fluorescence intensity (MFI). Both fluorescence microscopy and flow cytometry analyses revealed that CRSJG effectively mitigated mito-Ca²⁺ aggregation in MPP⁺ induced nerve cells (Figures 4A–E), while maintaining cyto-Ca²⁺ levels at a reduced level (Figures 4F–J). The trends observed in mito-Ca²⁺ and cyto-Ca²⁺ levels following CRSJG intervention paralleled those seen with 2-APB, an inhibitor of intracellular Ca²⁺ transport, albeit with less pronounced effects than 2-APB.

We assessed ROS level and MMP in MPP⁺ induced nerve cells using flow cytometry. ROS level in these cells were significantly elevated due to MPP⁺ induction but were effectively normalized upon CRSJG treatment (Figures 5A–C). Concurrently, both CRSJG and 2-APB significantly mitigated the reduction in MMP caused by MPP⁺ (Figures 5D,E).

The rates of late-apoptosis and total-apoptosis in MPP⁺ induced nerve cells were significantly elevated, with interventions using CRSJG and 2-APB effectively mitigating this MPP⁺ induced apoptosis (Figures 6A–C). Additionally, Western blot analyses indicated that CRSJG and 2-APB substantially decreased the MPP⁺ induced upregulation of apoptosis-related proteins (Figures 6D–J). These findings demonstrate that CRSJG possesses a protective effect against MPP⁺ induced neuronal apoptosis.

Utilizing transmission electron microscopy, we examined the microstructure of nerve cells to observe alterations in MAMs. MPP⁺ induced substantial damage to MAMs structure within nerve cells, characterized predominantly by mitochondrial swelling, fragmentation or complete loss of mitochondrial cristae, and densely surrounded mitochondria by the endoplasmic reticulum, leading to tighter connections within MAMs. Intervention with CRSJG and 2-APB partially restored the mitochondrial and endoplasmic reticulum structures, and significantly expanded the MAMs (Figure 7A).

To further elucidate the functional changes in MAMs, Western blot analysis was employed to assess the expression of key proteins within the Ca²⁺ transport complex. Expressions of IP₃R, VDAC, MCU, and Grp75 were notably elevated in the MPP⁺ group, whereas interventions with CRSJG and 2-APB reduced these protein levels (Figures 7B–F). These findings suggest that CRSJG effectively ameliorates both the structure and function of MAMs in MPP⁺ induced nerve cell damage.