Biological evaluation of sophoridine by interaction with plasma transport protein and protective effects in cardiomyocytes

2.1 Materials

Alpha-2-macroglobulin from human plasma (≥95 %, SDS-PAGE, Cat No: SRP6314), and 1-anilinonaphthalene-8-sulfonate (ANS) were purchased from Sigma (USA). Sophoridine (≥98.0 %, Cat. No.: CS-0016780) from ChemScene LLC (USA). All other materials were of analytical grade and used without further purification.

2.2 Fluorescence spectroscopy

The fluorescence spectroscopy was done using a Shimadzu RF-5301 spectrophotometer (Tokyo, Japan). Intrinsic fluorescence spectroscopy measurements were carried out through exciting the α2M solution at a wavelength of 280 nm and reading the emission spectra at a wavelength range of 300–450 nm. The slit width for excitation and emission was set at 10 and 5 nm, respectively. The α2M concentration was fixed at 10 µM. The protein sample was then incubated with increasing concentrations of sophoridine (10–70 µM) for 2 min. Then the intrinsic fluorescence spectra were determined at four temperatures of 298, 303, 307, and 315 K. The intrinsic emission spectrum of sophoridine alone was also detected to exclude the interference of protein fluorescence signal by ligand. ANS fluorescence spectra were recorded to examine the effect of sophoridine on the surface hydrophobicity changes in α₂M. The interaction of α2M/ANS (10/100 µM) and sophoridine (70 µM) was explored by ANS fluorescence spectroscopy at room temperature. The samples were excited at 360 nm and excitation was read in the range of 400–600 nm. All spectra were corrected against inner filter effect as previously described (El Gammal et al., 2022).

2.3 Far-UV circular dichroism (CD)

Far-UV CD spectra measurements were done using a JASCO J-1500 spectropolarimeter. CD spectra of α₂M (20 μM) and α₂M-morin mixture (70 μM) were recorded at room temperature. Each spectrum was recorded from the average of three scans with a scanning speed of 500 nm/min. All CD spectra were corrected against baseline spectra of buffer and ligand chirality.

2.4 Molecular docking

Molecular docking analysis of α₂M with sophoridine was performed using AutoDock Vina software. The 3D crystal structures of α₂M (PDB ID: 6TAV) and sophoridine (PubChem CID: 165549) were obtained from the RCBS Protein Data Bank and PubChem, respectively. AutoDock tools were used to perform the α₂M and sophoridine preparation, which were removing bound ligand and water molecules, and adding Kollman charges and hydrogen atoms. To cover the α₂M structure, the grid center was modified to X = 15.38, Y = 138.4, Z = 56.36 with a grid-box dimension of X = 82, Y = 126, Z = 126.

2.5 Cell culture

H9c2 cardiomyocyte cells obtained from the American Type Culture Collection (ATCC, USA.) were cultured in DMEM (Gibco, USA) containing 10 % fetal calf serum (FCS, Gibco, USA) in a humidified incubator with 5 % CO₂ at 37 °C. The medium was renewed every 2–3 days, and cells were used for experimental studies at 80–90 % confluence.

2.6 Myocardial ischemia/reperfusion (MI/R) injury model in vitro

To induce the MI/R injury in vitro, this experiment was done using the method previously established (Esumi et al., 1991) and reused in Yin et al. paper (Yin et al., 2013). Briefly, H9c2 cardiomyocyte cells were exposed to an ischemic buffer (Yin et al., 2013) in a hypoxic/ischemic chamber (Anaerocult® A mini, Merck) for 2 h in a humidified incubator with 5 % CO₂ and 95 % nitrogen at 37 °C. To perform reperfusion, the cells were cultured in fresh medium in the presence or absence of sophoridine (20 μM) for 2 h at 95 % O₂ and 5 % CO₂.

2.7 Cell viability analysis

The vehicle and treated H9C2 cells were used for cell viability study using MTT assay (Yin et al., 2013). Briefly, 20 μl of MTT solution (5 mg/ml) was added to each well (×10⁴ cells/well) followed by incubation for 4 h at 37 °C, aspiration of supernatants, and adding 150 μl DMSO. After 15 min, the optical density was read at 490 nm using a Microplate Reader (Synergy-HT, BioTek, USA).

2.8 Measurement of lactate dehydrogenase (LDH) release

To membrane damage study was assessed using the Lactate Dehydrogenase Activity Assay Kit (MAK066, Sigma, USA). After experimental treatment, the culture medium of cells was removed and used for LDH activity according to the manufacturer's protocol at 450 nm on a Microplate Reader (Synergy-HT, BioTek, USA).

2.9 Measurement of reactive oxygen species (ROS)

Cells were washed with PBS and then incubated with 1 ml working solution of 2,7-dichlorofluorescin diacetate (DCFH-DA, Sigma, USA) at 37 °C for 30 min. Then, the cells were washed with PBS, lysed, and centrifuged at 2300g for 10 min (Ahamed et al., 2013). The fluorescence intensity of dichlorofluorescein (DCF) in the supernatant (200 μl) was read at 485 nm excitation and 520 nm emission using a Microplate Reader (Synergy-HT, BioTek, USA).

2.10 Oxidative stress assay

The vehicle and treated H9C2 cells were used for oxidative stress assays using commercial Kits, malondialdehyde (MDA, cat. no. ab118970), and catalase (CAT, cat. no. ab83464) according to the manufacturer's protocol.

2.11 Enzymatic caspase-9, -3 assay

The caspase activity was measured using the manufacturer's protocol (Promega Corporation, Madison, WI, USA) (Yin et al., 2013). Briefly, cells were harvested, washed, lysed, and centrifuged at 14,000 rpm for 15 min. Then, protein concentration was determined by Bradford Kit (Sigma, USA) and a fixed protein concentration was incubated with caspase assay buffer (30 μl) as well as caspase substrates (2 μl) at 37 °C for 4 h. The caspase activity was measured at 405 nm on a Microplate Reader (Synergy-HT, BioTek, USA).

2.12 Quantitative real-time PCR (qPCR)

All cells were washed with PBS several times and then RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to extract RNA following the manufacturer's protocol. Then, cDNA was synthesized by reverse transcription reaction with QuantiTect Reverse Transcription Kit (TaKaRa, Dalian, China). DNA was amplified with selective primers (Bax, Bcl-2, caspase-9, caspase-3) and the internal control gene (GAPDH) (Fang et al., 2018). SyberGreen qPCR Mix (TaKaRa, Dalian, China) was used for qPCR following the manufacturer's protocol. Real-Time PCR experiment was done using an ABI-step I plus (Applied Biosystems, Forster City, CA, USA) instrument. The relative expression was done through standardization of the expression of target genes by GAPDH and expressed as fold change using the 2^−ΔΔCt method.

2.13 ELISA analysis

After treatment, the cells were collected, lyzed, and centrifuged at 4 °C for 20 min at 10,000 rpm (Yin et al., 2013). After the determination of protein concentration by Bradford assay, 30 μg of protein was used for the assessment of human phosphorylated type of PI3K (p-PI3K, ELISA Kit, Cat. No: MBS167579, MyBioSource, Inc. USA) and p-Akt (ELISA Kit Cat. No: MBS9518725, MyBioSource, Inc. USA) following the manufacturer's protocol.

2.14 Statistical analysis

SPSS was used to perform statistical analysis. Data were reported as the mean ± standard deviation (SD) of three experiments. The statistical comparisons were done with an analysis of variance (ANOVA) followed by a post hoc Tukey's test. P < 0.05 was considered to show a statistically significant difference.

3.1 Fluorescence quenching study

The intrinsic fluorescence signal of α2M was measured and then compared to the fluorescence spectra of the α2M-sophoridine complexes to measure structural changes of proteins induced by the ligand (Rehman et al., 2017). The effect of ligand (sophoridine) on α2M intrinsic fluorescence intensity is exhibited in Fig. 1A. The addition of sophoridine to α2M led to a decrease in the intrinsic fluorescence signal of α2M evidenced by fluorescence spectra. This finding infers the probable microenvironmental changes around the α2M chromophore (Zia et al., 2024). Also, as shown in Fig. 1A, the maximum wavelength of α2M was red-shifted from around 340–335 nm upon interaction with 70 µM sophoridine. This small red shift in maximum emission wavelength corroborates with the possible conformational changes around chromophores, Trp and Tyr residues (Zia et al., 2019; Siddiqui et al., 2020).

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Fig. 1

(A) Interaction of α2M and sophoridine explored by fluorescence spectroscopy at room temperature. The α2M concentration was 10 µM and added by increasing concentrations of sophoridine (10–70 µM). (B) Stern-Volmer plots of the α2M-sophoridine complex at different temperatures. (C) Modified Stern-Volmer plots of the α2M-sophoridine complex at different temperatures. (D) van Hoff's plot of the α2M-sophoridine complex at different temperatures.

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3.2 Fluorescence quenching mechanism

The fluorescence quenching mechanism could be categorized as dynamic, static, or both static-dynamic for the interaction of a ligand–protein (Katrahalli et al., 2023; Rehman et al., 2024; Shamsi et al., 2024). Dynamic and static-type quenching refers to collisions and ground-state complex formation between the receptor and quencher, respectively (Agrawal et al., 2019; Rehman et al., 2024). By analyzing fluorescence quenching data, the probable type of fluorescence quenching mechanism can be determined using Stern–Volmer Eq. (1) as follows (Rehman et al., 2024):

It can be seen that there is a direct relationship between K_SV values and temperature, revealing that the fluorescence quenching type of α2M by sophoridine occurs through a dynamic mechanism (Bekhouche et al., 2012). However, the k_q values for the fluorescence quenching of α2M by sophoridine exceeds the threshold of biomolecule's limiting diffusion constant (2.0 × 10¹⁰ dm³ mol⁻¹ s⁻¹), supporting the existence of a static mechanism (Manivel et al., 2023). Therefore, it can be claimed that both static-dynamic mechanisms meditate the decrease in fluorescence intensity of α2M by sophoridine.

3.3 Binding affinity

If every ligand independently interacts with a binding site on a protein, the binding affinity for a protein–ligand system can be calculated using Eq. (2) given below [28]:

One binding site was determined for sophoridine on α2M evidenced by n values, which are around 1. Upon increasing temperature, the n values decrease, likely due to the change in the population of ligand molecules in the ground state at higher temperatures (Gokavi et al., 2023). The calculated K_b values were around 10⁴, demonstrating a favorable (moderate) interaction between sophoridine and α2M. It was also found that the K_b values are higher in 315 K compared to 298 K, revealing the greater stability of the α2M-sophoridine complex at higher temperatures stemmed from the involvement of hydrophobic, hydrogen bonding, and electrostatic interactions, which will be further studied in the next section.

3.4 Thermodynamic analysis

The potential forces involved in the interaction of sophoridine and α2M could be determined from the values of enthalpy change (ΔH⁰) and entropy change (ΔS⁰) which can be determined using van Hoff's Eq. (3) as follows (Gokavi et al., 2023):

Then, as demonstrated in Eq. (4), the free energy change (ΔG⁰) can be calculated by the Gibbs–Helmholtz relationship (Gokavi et al., 2023):

3.5 Structural changes studies

Results from ANS fluorescence spectroscopy measurement provided detailed information for the interaction of sophoridine and α₂M. The surface hydrophobicity of the protein is the most frequently marker employed in research on protein conformational changes (Alizadeh-Pasdar and Li-Chan 2000, Wang et al., 2024). Because of its strong affinity for interacting with nonpolar residues, the ANS fluorescence intensity measurement offers detailed insight for examining the exposure of some hydrophobic protein patches (Wang et al., 2024). The measured ANS fluorescence intensity is heavily dependent on the hydrophobic properties of a protein (Li et al., 2024). The ANS fluorescence spectra of α₂M in the presence and absence of sophoridine were recorded and displayed in Fig. 2A. The ANS fluorescence spectrum of α₂M was detected to slightly increase at 484 nm upon interaction with 70 μm of sophoridine, suggesting the partial exposure of hydrophobic residues (Li et al., 2024). As a result, the conformational changes of α₂M through exposure of some hydrophobic groups caused by interaction with sophoridine.

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Fig. 2

(A) Interaction of α2M (10 µM) and sophoridine (70 µM) explored by ANS fluorescence spectroscopy at room temperature. (B) Interaction of α2M (20 µM) and sophoridine (70 µM) explored by Circular dichroism study.

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The interaction of small molecules with proteins could also induce significant structural changes in proteins (Ansari et al., 2023a,b). Indeed, the secondary structure of protein is a crucial aspect of protein stability and relevant function (Zeeshan et al., 2019). The changes in the protein secondary conformer upon ligand binding is typically analyzed using far-UV CD spectrum (190–250 nm) (Sreerama et al., 2000). The far-UV CD spectrum of free α₂M depicts a minimum at 217 nm (Fig. 2D), which is characteristics of the dominance of β-sheet structure (Ansari et al., 2023a,b; Ansari et al., 2024). Upon the presence of sophoridine, the intensity (chirality) of the minimum (217 nm) slightly reduced (Fig. 2D), suggesting the partial changes in the secondary structure of α₂M (Ansari et al., 2024). In other words, following incubation with sophoridine, no significant far-UV CD peak shifting or changes were observed, which indicated a partial decrease in β-sheet structure content of α₂M (Zia et al., 2022; Ansari et al., 2023a,b).

3.6 Molecular docking study

Molecular docking as a theoretical approach is widely used to study the interactions between ligands and proteins. By this method, we can obtain detailed information about the location of the ligands in the protein binding site (Fu et al., 2018). Based on the unique structure adopted by proteins in the biological media, a small molecule can bind to different proteins in various modes depending on the hydrogen bond pairing and surface shape complementarity (Luo et al., 2010). In the present study, in the docking analysis, the energetically most favorable docked site of sophoridine and α2M with a binding score of −6.93 kcal mol⁻¹ is shown in Fig. 3A. Further analysis indicated that sophoridine exhibits several hydrophobic and hydrogen bond interactions with α2M (Fig. 3B, Table 4). The binding site analysis demonstrated hydrogen bonding interaction between sophoridine and α2M amino acid residues at the sites of GLU737, PHE735, PRO736, THR738, TRP739 (Fig. 3B). Moreover, sophoridine interacted with VAL758, PHE735, PHE735, and TRP739 amino acid residues through hydrophobic forces (Alkyl, Pi-Alkyl, Pi-Alkyl, Pi-Alkyl) (Fig. 3B).

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Fig. 3

(A) Interaction of α2M and sophoridine explored by molecular docking study. (B) Amino acid residues in the binding pocket.

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Experimental data showed that the interaction of sophoridine with α2M led to fluorescence quenching. A molecular docking study reveals that TRP739 amino acid residue is placed in the docked pose of the α2M-sophoridine complex. Hence, the finding of the molecular docking study in agreement with experimental data imply that α₂M molecule has a favorable affinity for sophoridine, and hydrophobic interactions and hydrogen bonds are the most contributing forces in the complexation process.

3.7 Sophoridine protects cardiomyocytes against MI/R injury in vitro

H9c2 cardiomyocyte viability after MI/R was studied by MTT assay (Fig. 4A). When compared to the control cells, it was shown that MI/R significantly reduced cardiomyocyte cell viability to 62.32 ± 2.94 % (**P < 0.01), however sophoridine therapy (20 μM) significantly increased cell viability to 87.97 ± 13.57 %. This data pointed out that cell survival was considerably restored by the treatment of cells with sophoridine.

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Fig. 4

After the induction of MI/R injury in vitro, the protective effects of sophoridine (20 μM) for 2 h on cell viability and membrane leakage were assessed by (A) MTT assay and (B) LDH assay. **P < 0.01. The data are presented as mean ± SD of three experiments.

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Furthermore, cytoplasmic enzyme amount in cell culture medium as a marker of cell membrane damage showed that LDH release notably increased to 172.39 ± 16.31 % after MI/R compared to the control group (P < 0.01) (Fig. 4B). However, sophoridine treatment led to a decrease in LDH release to 124.42 ± 9.84 % (P < 0.01) at the end of reperfusion. This data is in good agreement with previous studies indicating that several kinds of naturally occurring compounds such as coptisine (Wang et al., 2017), matrine (Guo et al., 2018), oxymatrine (Zhang et al., 2021), and sinomenine (Xia et al., 2022) could confe0072 protection against MI/R injury by mitigation of membrane damage.

3.8 Sophoridine protected cardiomyocytes from oxidative stress

Numerous studies have shown that regulating oxidative stress is a potential strategy that bioactive materials can trigger their protective effects against MI/R injury (Yu et al., 2016; Shu et al., 2019; He et al., 2024). Therefore, in this study, we aimed to assess the oxidative stress markers such as ROS, MDA and CAT in H9C2 cells. The data showed that the ROS (Fig. 5A) and MDA (Fig. 5A) levels were remarkably elevated by MI/R injury; however, these contents were reduced following treatment with sophoridine. The CAT activity in cardiomyocytes subjected to MI/R injury was significantly inhibited relative to the control group (Fig. 5C). However, sophoridine notably recovered the CAT activity in MI/R-triggered H9C2 cardiomyocytes (Fig. 5C).

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Fig. 5

After the induction of MI/R injury in vitro, the protective effects of sophoridine (20 μM) for 2 h on oxidative stress were assessed by (A) ROS assay, (B) MDA level, (C) CAT activity assay. *P < 0.05, **P < 0.01, ***P < 0.001. The data are presented as mean ± SD of three experiments.

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Xia et al. reported that sinomenine as an alkaloid obtained from Sinomenium acutum could show protective effects against MI/R injury by mitigation of oxidative stress-mediated by inhibition of ROS generation and MDA formation (Xia et al., 2022). Therefore as mechanistic insight, it can be suggested that alkaloids could modulate the cardiomyocyte cytotoxicity stemming from MI/R injury through attenuating oxidative stress via upregulation of antioxidant enzymes as well as reduction of ROS and MDA formation (Qi et al., 2024).

3.9 Sophoridine protected cardiomyocytes from apoptosis

The intrinsic pathway of apoptosis is hardly regulated by mitochondria mediated by the regulation of the Bcl-2 family (Dadsena et al., 2021; Wolf et al., 2022). While Bcl-2 acts as an anti-apoptotic effector, Bax is a pro-apoptotic marker (Czabotar and Garcia-Saez 2023). Upregulation of Bcl-2 and downregulation of Bax have been previously shown to promote the anti-apoptotic effects of bioactive compounds in cardiomyocytes against MI/R injury (Yu et al., 2016; Su et al., 2022; Ge et al., 2023). Also, mitochondrial apoptosome-driven caspase upregulation has been known as an important mechanism triggered by cytotoxic effects and leads to the activation of several caspases in apoptosis (Creagh et al., 2003). The activation of caspases-3 and -9, however, has been exhibited to serve as the most important stimulatory factor for the induction of apoptosis during MI/R injury (Stephanou et al., 2001). Therefore, protecting cardiomyocytes from MI/R injury could be mediated by blocking caspase-3 and caspase-9 expression and activity during apoptosis (Liu et al., 2020).

To explore how sophoridine protects cardiomyocytes against cytotoxicity induced by MI/R injury in vitro, apoptosis markers via the intrinsic pathway, expression of Bcl-2, Bax, caspase-9, and caspase-3 mRNA along with caspase-9 and caspase-3 activity assays in H9c2 cardiomyocyte cells were studied. As shown in Fig. 6A, B, MI/R significantly upregulated the relative mRNA level of pro-apoptotic proteins, Bax, caspase-9, and caspase-3, which was associated with the reduction of the anti-apoptotic protein, Bcl-2, relative to the control group. However, this mechanism was suppressed by incubation of cardiomyocytes with sophoridine. In addition, similar trends with caspase mRNA assays were observed in caspase activity studies (Fig. 6C).

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Fig. 6

After the induction of MI/R injury in vitro, the protective effects of sophoridine (20 μM) for 2 h against apoptosis were assessed by (A) qPCR assay for expression of Bax and Bcl-2 genes, (B qPCR assay for expression of caspase-9 and caspase-3 genes, (C) caspase-9 and caspase-3 activity assay. **P < 0.01, ***P < 0.001. The data are presented as mean ± SD of three experiments.

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These data revealed that sophoridine was able to inhibit the MI/R-initiated upregulation of apoptosis-associated genes, which is consistent with previous studies, describing the protective effects of natural compounds against MI/R injuries (Yin et al., 2013; Yu et al., 2016; Han et al., 2019). For example, Xing et al. reported that anisodamine as a natural alkaloid drug could show a potential cardioprotective effect against MI/R through regulation of apoptosis mediated by downregulation of Bax/Bcl-2 protein ratio and expression of caspases (Xing et al., 2015).

3.10 Sophoridine activates PI3K/AKT signaling pathway

One important signaling cascade that regulates apoptosis is PI3K/AKT. The stimulation of the PI3K/AKT signaling pathway downregulates the expression levels of caspase-3 and -9 (Ghafouri-Fard et al., 2022). Previous studies have mentioned that bioactive products are capable of inhibiting MI/R-stimulated cardiomyocyte apoptosis mediated by targeting PI3K/AKT (Syed Abd Halim et al., 2023). For example, Li et al. documented that piperine alkaloid, the primary component of the grains of black pepper, protects against MI/R injury mediated by upregulating the PI3K/AKT signaling pathway (Li et al., 2021). The same effects have been also reported for berberine which is able to attenuate MI/R injury via activation of the PI3K/AKT signaling pathway (Chen et al., 2014). Therefore, in this study, we assessed the activation of the PI3K/AKT signaling pathway in H9C2 cardiomyocytes exposed to MI/R in the absence and presence of sophoridine by ELISA assay. As shown in Fig. 7, MI/R markedly decreased the expression of p-PI3K and p-AKT in comparison with the control group. However, treatment of cells with sophoridine at the induction of MI/R upregulated the expression of p-PI3K and p-AKT compared with the MI/R-treated group.

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Fig. 7

After the induction of MI/R injury in vitro, the protective effects of sophoridine (20 μM) for 2 h on the regulation of AKT/PI3K signaling pathways were assessed by ELISA. **P < 0.01, ***P < 0.001. The data are presented as mean ± SD of three experiments.

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Therefore, it can be surmised that MI/R injury was ameliorated by sophoridine, likely via activation of the PI3K/AKT signaling pathway. This data is consistent with previous studies, reporting the protective effects of bioactive compounds, particularly alkaloids, against MI/R injury via the upregulation of the PI3K/AKT signaling pathway (Chen et al., 2014; Li et al., 2021; Syed Abd Halim et al., 2023).