Gamma globulin binding of hinokiflavone with anti-osteoporosis effects: A mechanistic study

2.1 Materials

Hinokiflavone (98 %, HPLC) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). γ-Globulin, Fetal bovine serum (FBS), α-modified minimal essential medium (α-MEM), dichlorodihydrofluorescein diacetate (DCFH-DA), and N-acetyl-l-cysteine (NAC) were purchased from Sigma Chemical (St. Louis, MO, USA). The osteoblastic MC3T3-E1 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Other materials were of high grade and purchased from Sigma Chemical (St. Louis, MO, USA). The HNK stock solution (1 mM) was dissolved in dimethyl sulphoxide (DMSO) and was further diluted with sodium phosphate buffer (50 mM, pH 7.5). The γ-globulin solution was prepared in sodium phosphate buffer (50 mM, pH 7.5).

2.2 Spectroscopy measurements

The intrinsic fluorescence measurements were done on an F-2500 spectrophotometer (Hitachi, Japan), where the excitation wavelength was set at 280 nm and the emission wavelengths were set between the ranges of 300–420 nm. To explore the interaction of HNK and γ-globulin, 2.0 mL of γ-globulin solution (5 µM) was titrated with consecutive addition of HNK (1–30 µM). The experiments were performed at the four different temperatures of 298, 305, 310, and 315 K. All fluorescence data were corrected against HNK intrinsic fluorescence and inner filter effects (Zia et al., 2024). Synchronize fluorescence spectroscopy (SFS) study for determination of the microenvironmental changes of Tyr and Trp residues of γ-globulin was done at Δλ between excitation and emission wavelength at 15 nm and 60 nm, respectively.

2.3 Molecular docking study

To understand the molecular interaction of HNK with γ-globulin at the atomic level, a molecular docking study was done using AutoDock Vina package. The 3D molecular building of HNK was downloaded from PubChem (PubChem CID: 528444) (https://pubchem.ncbi.nlm.nih.gov/). The γ-globulin crystal structure (PDB code: 1AJ7) was obtained from Protein Data Bank (https://www.rcsb.org/). AutoDock Vina Tool was used to control the hydrogen atoms and charges as well as the elimination of water molecules. A blind docking study was performed at a box of 28 × 28 × 28 Å, and then the lowest interaction score was selected for further analysis.

2.4 Cell culture

The osteoblastic MC3T3-E1 cells were cultured in a-MEM supplemented with 10 % heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin with 5 % CO₂ at 37 °C.

2.5 Cell viability assay

The cells were incubated with H₂O₂ with a concentration of 100 µM for different times of 6 h, 12 h, 24 h, 48 h, and 72 h to induce oxidative stress. To measure the cytotoxicity of HNK, the cells were treated with different concentrations of HNK (1–50 µM) for 72 h. To investigate the protective effects of HNK on H₂O₂-induced cytotoxicity in MC3T3-E1 cells, the cells were incubated with H₂O₂ (100 µM) and H₂O₂ + HNK (20 μM) for 72 h. NAC with a concentration of 1 mM was also used as the positive control for all assays. Then the cell viability assay was done using the MTT assay as described previously at 570 nm absorbance using an ELISA reader (Thermo Scientific, MA, USA) (Park et al., 2020).

2.6 Alkaline phosphatase (ALP) activity

Alkaline phosphatase (ALP) activity was done based on the previous study (Zhang, Yang et al., 2012). Briefly, following treating the cells in an osteogenic medium for 6 days, the cells were cultured with serum-free medium containing H₂O₂ (100 µM), HNK (20 μM), H₂O₂ (100 µM) + HNK (20 μM), or H₂O₂ + NAC (1 mM) for 72 h. Then, the cells were harvested, washed, lysed, and centrifuged at 10,000g for 5 min. The supernatant was then used to assess the ALP activity using the relevant kit (MAK447, Sigma) according to the manufacturer's protocol.

2.7 Quantitative real-time PCR (qPCR)

The cells cultured in an osteogenic medium for 6 days were exposed to H₂O₂ (100 µM), HNK (20 μM), H₂O₂ (100 µM) + HNK (20 μM), or H₂O₂ + NAC (1 mM) for 72 h. Total cellular RNA was then extracted using the Trizol reagent according to the manufacturer's instructions (TaKara, Dalian, China). Single-strand cDNA synthesis was synthesized by the PrimeScript RT reagent kit (TaKaRa, Dalian, China). Real-time PCR was performed using a SYBR® Premix Ex Taq™ (Takara, Dalian, China). qPCR was performed on an ABI PRISM 7700 sequence detection system (Applied Biosystems, Grand Island, NY, USA).

The primer sequences and experiment procedure for ALP, collagen-I, Bax, Bcl-2, Caspase-3, and β-actin were developed based on the previous studies (Zhang et al., 2012; Guo et al., 2014).

2.8 ELISA assay

ELISA kits for the quantitative determination of Bax, Bcl-2, caspase-3, and collagen-I were purchased from Cusabio Biotechnology (Wuhan, China). The cells were cultured in an osteogenic medium for 6 days. Then, the cells cultured with serum-free medium containing H₂O₂ (100 µM), HNK (20 μM), H₂O₂ (100 µM) + HNK (20 μM), or H₂O₂ + NAC (1 mM) for 72 h were harvested, washed, lysed, and centrifuged at 10,000g for 5 min. The supernatant was then used to perform ELISA assay according to the manufacturer's protocol.

2.9 Measurement of intracellular ROS

The hydrolysis of the non-fluorescent DCFH-DA probe can be catalyzed by intracellular esterase and intracellular ROS to produce fluorescent dichlorofluorescein (DCF). The measurement of intracellular ROS was done based on a previous study (Yu et al., 2013). In brief, the cells were The cells incubated with H₂O₂ (100 µM), HNK (20 μM), H₂O₂ (100 µM) + HNK (20 μM), or H₂O₂ + NAC (1 mM) for 72 h were rinsed with PBS, incubated with 10 μM DCFH-DA for 30 min at 37 °C, harvested, and washed. Then, ROS production was assessed by reading the fluorescence intensity of cells at an excitation wavelength of 488 nm and an emission wavelength of 525 nm on a fluorescence microplate reader (Victor® X3, Perkin Elmer, Waltham, MA, USA).

2.10 Measurement of membrane lipid peroxidation

The extent of lipid peroxidation was determined using the MDA assay Kit (Beyotime Institute of Biotechnology (Shanghai, China) according to the manufacturer's protocol. Briefly, the cells were treated with H₂O₂ (100 µM), HNK (20 μM), H₂O₂ (100 µM) + HNK (20 μM), or H₂O₂ + NAC (1 mM). After 72 h, the cell samples were mixed with a working solution (200 μl MDA assay), heated at 100 °C for 15 min, cooled, and centrifuged at 1000 × g for 10 min. The optical density of supernatants was read at 532 nm using an ELISA reader (Thermo Scientific, MA, USA).

2.11 Statistical analysis

The data in cell culture assays were from three replicates and the data were expressed as the mean ± standard deviation (S.D.). The data were analyzed using one-way ANOVA using SPSS 22.0 software (Chicago, IL, USA). A statistically significant difference was considered at p < 0.05.

3.1 Spectroscopic studies

Quenching of the aromatic residue fluorescence intensity of γ-globulin was detected following consecutive addition of HNK at different temperatures (Fig. 1A). The fluorescence spectrum of HNK at an excitation at 280 nm was subtracted from protein fluorescence signal to ensure that the fluorescence intensity of HNK does not play a significant role in the quenching experiment. The addition of HNK to γ-globulin was observed to not only induce a fluorescence quenching of protein but also change the λmax of aromatic residue (redshift). This phenomenon can be associated with the changes in the surrounding polarity of aromatic residues in γ-globulin upon interaction with HNK.

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Fig. 1

(A) The interaction of γ-globulin with consecutive addition of HNK measured by fluorescence quenching study at 298 K. (B) The modified Stern-Volmer plots of γ-globulin-HNK system at four different temperatures. (C) The Stern-Volmer plots of γ-globulin-HNK system at four different temperatures. (D) The van’t Hoff plots of γ-globulin-HNK system at four different temperatures. SFS measurement of γ-globulin-HNK system at (E) Δλ = 60 nm and (F) Δλ = 15 nm.

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It can be indicated that the surrounding polarity of the aromatic residue in γ-globulin was changed by HNK, which has already been reported for the interaction of some other polyphenols with this protein (Li et al., 2021).

Binding parameters for interaction between HNK and γ-globulin were determined using a double logarithmic equation as follows (Sharma et al., 2022):

The resultant plots derived from Eq. (1) are shown in Fig. 1B. K_b is indicative of the stability of the formed protein–ligand complex. The in vitro K_b and n values between γ-globulin and HNK were found in the order 10⁴ M⁻¹ and 1, respectively, suggesting a moderate probable binding with one binding site between HNK and γ-globulin in vivo (Li et al., 2023). In line with this study, Li et al. reported that the formation of baicalin–γ-globulin, quercetin–γ-globulin, myricetin–γ-globulin, rutine–γ-globulin, diadzein–γ-globulin, hesperidin–γ-globulin, and isoliquiritin–γ-globulin complexes occurs with K_b values of around 10⁴ (Li et al., 2021).

Also, it was detected that the K_b values of HNK–γ-globulin declined with increasing temperature (Fig. 1B, Table 1), implying that the covalent forces are not predominated in the HNK–γ-globulin complex formation, while noncovalent forces are mainly involved and these interactions become stronger at lower temperature compared to higher temperatures.

A similar temperature effect was also observed for the K_b values of baicalin–γ-globulin, quercetin–γ-globulin, rutin–γ-globulin, daidzein–γ-globulin, and hesperidin–γ-globulin complexes, while myricetin–γ-globulin and isoliquiritin–γ-globulin complexes behaved differently (Li et al., 2021). As a result, affinities of flavonoids for protein binding were heavily affected by their structural differences (Yang et al., 2011). Bioconjugate between γ-globulin and HNK as a C–O–C-type biflavonoids was found to have similar stability with the complex between γ-globulin and several classic flavonoids. This indicates a potential complexation between γ-globulin and HNK. Also, it was detected upon an increase in temperature, the n values decreased slightly because of subtle structural changes at the binding site of the γ-globulin.

Quenching constant (K_SV) was computed from the Stern–Volmer (SV) Eq. as follows (Sharma et al., 2022):

SV plots for the HNK–γ-globulin complex at three different temperatures are exhibited in Fig. 1C and the calculated K_SV values are listed in Table 2.

The K_SV values give information regarding the fluorescence quenching efficiency of protein by ligand and the variation of these values with temperature indicates the static or dynamic quenching mechanisms.

The higher value of K_SV for HNK (10⁵ M⁻¹) compared to baicalin, quercetin, myricetin, rutin, diadzein, hesperidin, and isoliquiritin (10⁴ M⁻¹) and puerarin (10³ M⁻¹) (Li et al., 2021) indicated that this C-O-C-type biflavonoid quenched the aromatic residue fluorescence of γ-globulin more efficiently than the classical flavonoids (Table 1). A rise in temperature increased the quenching process of γ-globulin by HNK. A similar phenomenon was already reported during the fluorescence quenching of γ-globulin by most classical flavonoids, which suggests a dynamic quenching mechanism (Yang, Zhao et al., 2011; Li, Wang et al., 2021). Also, the determination of the quenching process was done using the k_q values as calculated from Eq. (2).

The calculated values of k_q were determined as ∼10¹³ M⁻¹ s⁻¹, which was significantly higher than the maximum reported value of k_q (10¹⁰ M⁻¹ s⁻¹) for dynamic quenching (Sharma et al., 2022). Accordingly, it can be implied that both static and dynamic mechanisms of quenching are involved in this system, which was similar to the quenching by other classical flavonoids (Li et al., 2021). Li et al. suggested that the possible quenching mechanism may be initiated by static quenching and is further followed by dynamic one (Li et al., 2021).

Different intermolecular forces typically contribute to the protein–ligand complexation process. The sign and magnitude of the enthalpy change (ΔH) and entropy change (ΔS) are usually used to evaluate the nature of the binding forces (Abarova et al., 2024; Chaves et al., 2024). ΔS and ΔH values along with Gibbs free energy (ΔG) parameter were then determined by the van’t Hoff Eq. (3) and Gibbs Eq. (4) as follows (Chaves et al., 2024):

Fig. 1D shows the van’t Hoff plot for the interaction of HNK and γ-globulin at four different temperatures. The thermodynamic parameters were then listed in Table 3.

A negative ΔG value emphasizes that the HNK–γ-globulin complexation process occurred spontaneously. The values of ΔH and ΔS for the interaction of HNK and γ-globulin revealed that this process was based on an enthalpy driven system, indicating that hydrogen bond and van der Waals forces are predominant in the formation of such complex (Abarova et al., 2024; Chaves et al., 2024). An earlier report has shown that classical flavonoids such as baicalin, quercetin, myricetin, rutin, diadzein, hesperidin, isoliquiritin, and puerarin interact with γ-globulin through hydrophobic or electrostatic forces, while hydrogen bonds and van der Waals forces have played a minimum role (Li et al., 2021), revealing that HNK as a C–O–C-type biflavonoid interacts with γ-globulin differently from other classical flavonoids. Changes in the flavonoid structure from classical geometry to biflavonoid altered the nature of binding forces between the γ-globulin and HNK.

In this study, the conformation changes of γ-globulin induced by HNK were studied using SFS spectroscopy. The SFS technique is known as a very serviceable approach to obtaining information about the polarity changes in the vicinity of the Trp and Tyr residues. When Δλ values are fixed at 60 nm and 15 nm, this technique can give some details about the changes in the polarity of Trp and Tyr residues, respectively (He et al., 2015; Samandar et al., 2023). Fig. 1, exhibits the SFS analysis of γ-globulin in the presence of consecutive addition of HNK. It can be observed that when Δλ = 60 nm (Fig. 1E), a gradual quenching occurs in the fluorescence intensity accompanied by no shift, while when Δλ = 15 nm (Fig. 1F), an obvious red shift was observed. This implies that the polarity around Trp remains unchanged in the presence of HNK, while the red shift in the emission maxima of Try designates a reduction in the hydrophobicity surrounding the Tyr residues (Li et al., 2021; Sun et al., 2021).

Hence, these findings suggested that the interaction of γ-globulin with HNK does not alter substantially the polypeptide backbone and associated β-sheet structure of γ-globulin.

3.2 Molecular docking study

One of the most widely utilized approaches for investigating the binding affinity of ligands with protein is the analysis of molecular interaction using molecular docking study (Azimirad et al., 2023). In the present study, the interaction of the NHK (Fig. 2A) and γ-globulin was explored by using the AutoDock Vina package. The tertiary structure of γ-globulin is characterized by β-sheet structure (Fig. 2B). The major NHK-γ-globulin location is depicted in Fig. 2B and C, which exhibited that HNK as a ligand potentially binds to an interface of light and heavy chains. The docking energies for 10 runs were −44.768 to −30.543 kJ/mol, indicating a strong binding affinity between HNK and γ-globulin. The lowest binding energy conformation (−44.768 kJ/mol) was applied and the results showed that NHK could interact with several amino acid residues (Fig. 2D and E) mediated by different binding forces. It was shown that hydrogen bond: Ala 84 and Gln 39, weak hydrogen bond: Gly 106, and hydrophobic forces: Asp 85 and Phe 83 contributed to the formation of HNK-γ-globulin complex. This finding is in line with experimental results, which indicated that hydrogen bonding and van der Waals forces might play a role in the interaction of HNK and γ-globulin.

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Fig. 2

Molecular docking study of γ-globulin-HNK system. (A) HNK structure, (B) γ-globulin-HNK complex, (C) γ-globulin-HNK complex, (D) amino acids in the binding site, (E) amino acids in the binding site.

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Also, experimental assays showed that the interaction of HNK with γ-globulin leads to the fluorescence quenching of protein and slight conformational changes in the vicinity of Tyr residues. A molecular docking study showed that Tyr 87 (light chain) and Tyr 94 (heavy chain) amino acid residues are in the vicinity of the binding pocket of HNK on γ-globulin.

Both light and heavy chains are important binding sites on γ-globulin and typically a minimum number of small molecules show the possibility to bind there due to the extended structure (Wang et al., 2015; Krzyżak et al., 2023). The importance of these chains lies in their capability to interact with a number of small molecules, which makes them a notable candidate for drug delivery (Wang et al., 2015; Wang et al., 2016; Krzyżak et al., 2023). Also, the high affinity of γ-globulin to HNK not only causes their sequestration and reduced adverse effects but also changes the pharmacokinetics and pharmacodynamics of the HNK.

3.3 Protective effect of HNK on H₂O₂ stimulated cytotoxicity in MC3T3-E1 cells

Cell viability was done to assess the protective effect of HNK on the behavior of MC3T3-E1 cells against oxidative stress triggered by H₂O₂. First, we detected that H₂O₂ treatment at a concentration of 100 µM for 72 h induced the most significant cell death among other studied time intervals (Fig. 3A). We also found that the incubation of MC3T3-E1 with HNK at concentrations above 20 µM for 72 h induced significant cytotoxicity (Fig. 3B). Therefore, we treated the cells with H₂O₂ (100 µM) or H₂O₂ (100 µM) + HK (20 µM) for 72 h to assess the following experiments. As shown in Fig. 3C, when the cells were treated with HNK in the presence of H₂O₂, HNK could remarkably regulate the cell viability comparable to NAC as a potential antioxidant, suggesting that HNK might be able to suppress H₂O₂ triggered cytotoxicity.

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Fig. 3

(A) Effect of H₂O₂ on the viability of MC3T3-E1 cells at different time intervals. (B) Effect of HNK on viability of MC3T3-E1 cells at different concentrations after 72 h. (C) Effect of H₂O₂ (100 µM) + HK (20 µM) and H₂O₂ (100 µM) + NAC (1 mM) on viability of MC3T3-E1 cells after 72 h. The data were determined by MTT assay (SD, n = 3). *p < 0 0.05, **p < 0 0.01, ***p < 0 0.001, relative to control group.

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3.4 Protective effect of HNK on osteoblast dysfunction triggered by H₂O₂

In order to determine the protective effects of HNK on osteoblast dysfunction stimulated by H₂O₂, ALP activity was assessed. Compared with control cells, the addition of H₂O₂ significantly reduced ALP activity (Fig. 4A) and ALP mRNA expression (Fig. 4B) in MC3T3-E1 cells However, when osteoblasts were treated with HNK (20 µM) in the presence of H₂O₂ (100 μM), HNK significantly elevated ALP activity and ALP mRNA expression (Fig. 4A and B), which is known as one of the most important osteoblast differentiation markers (Khotib et al., 2023).

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Fig. 4

Effect of HNK (20 µM), H₂O₂ (100 µM), H₂O₂ (100 µM) + HK (20 µM) and H₂O₂ (100 µM) + NAC (1 mM) on (A) ALP activity (B) ALP mRNA expression, (C) collagen-I content, and (D) collagen-I mRNA of MC3T3-E1 cells after 72 h. *p < 0.05, **p < 0.01, ***p < 0.001, relative to control group.

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The evaluation of collagen-I contents at both protein (Fig. 4C) and mRNA (Fig. 4D) levels in MC3T3-E1 cells also indicated that when the cells were incubated with H₂O₂, collagen-I markers were reduced. However, when osteoblasts were treated with HNK (20 µM) in the presence of H₂O₂ (100 μM), HNK significantly elevated collagen-I protein and mRNA levels relative to H₂O₂-treated groups. Based on this data, in agreement with previous studies, we can discuss that biflavonoids similar to classical flavonoids could stimulate osteoblast differentiation through the regulation of ALP and collagen-I (Lee et al., 2006; Suh et al., 2013; Sekaran et al., 2022).

The increased collagen content and ALP activity are indicative of the osteo-stimulatory properties of HNK. It has been shown that apigenin as a building block of NHK can elevate MC3T3-E1 cell growth, collagen content and ALP activity at very low concentrations (Choi, 2007), which was comparable to the luteolin and hesperetin properties by restoration of collagen and ALP levels (Kim et al., 2011; Lin et al., 2020; Sekaran et al., 2022).

3.5 Protective effect of HNK on apoptosis induction triggered by H₂O₂

To determine the features of H₂O₂-induced death in MC3T3-E1 cells, ELISA and qPCR assays were carried out to evaluate the expression of Bax, Bcl-2, and caspase-3 as the typical markers of apoptosis. After treatment with H₂O₂for 72 h, the Bax protein and mRNA expression levels (Fig. 5A and B) were upregulated. However, when osteoblasts were treated with HNK (20 µM) in the presence of H₂O₂ (100 μM) for 72 h, HNK significantly decreased Bax protein and mRNA levels relative to H₂O₂-treated groups. Furthermore, it was observed that although the expression levels of Bcl-2 protein and mRNA were mitigated in the presence of H₂O₂, HNK remarkably upregulated the expression of Bcl-2 protein and mRNA levels relative to H₂O₂-treated groups (Fig. 5C and D).

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Fig. 5

Effect of HNK (20 µM), H₂O₂ (100 µM), H₂O₂ (100 µM) + HK (20 µM) and H₂O₂ (100 µM) + NAC (1 mM) on (A) Bax protein expression, (B) Bax mRNA expression, (C) Bcl-2 protein expression, (D) Bcl-2 mRNA expression, (E) caspase-3 protein expression, and (F) caspase-3 mRNA expression of MC3T3-E1 cells after 72 h. *p < 0.05, **p < 0.01, ***p < 0.001, relative to control group.

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We then assessed the antiapoptotic effects of HNK by determination of the caspase-3 content in MC3T3-E1 cells. Results demonstrated that H₂O₂ upregulated the expression of caspase-3 protein and mRNA (Fig. 5E and F). However, HNK was shown to reverse the H₂O₂-triggered apoptotic effect indicated through the downregulation of caspase-3 factor.

These results implied that HNK was capable of mitigating apoptosis induced by H₂O₂ in MC3T3-E1 cells. The HNK was able to inhibit H₂O₂-stimulated osteoblastic MC3T3-E1 cell apoptosis via ameliorating mitochondrial dysfunction evidenced by regulation of Bax, Bcl-2, and finally caspase-3. Bax and Bcl-2 play a key role in the mitochondrial integrity and deregulation of these factors can result in apoptosis induction through upregulation of caspase-3 (Adams and Cory, 2007; Asadi et al., 2022).

Osteoblast as the main bone cell type is the main driver of bone formation. A previous study has shown that exposure of MC3T3-E1 cells to 200 µM H₂O₂ can induce apoptosis and necrosis after 1 h (Fatokun et al., 2006). In this study, we found that 100 µM H₂O₂ can substantially reduce cell viability after 72 h, but the cytotoxicity effect was reduced significantly upon being treated with HNK. HNK has a polyphenolic structure similar to that of apigenin and these protective effects might be associated with the antioxidant characteristics of this compound.

3.6 Protective effect of HNK on oxidative stress induction triggered by H₂O₂

To investigate the antioxidant effects of HNK, the effects of HNK on H₂O₂-triggered ROS and MDA formation were examined. As expected, incubation with H₂O₂ for 72 h significantly increased ROS production (Fig. 6A) and MDA level (Fig. 6B). Co-treatment of H₂O₂ and NHK for 72 h significantly attenuated the effect of H₂O₂.

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Fig. 6

Effect of HNK (20 µM), H₂O₂ (100 µM), H₂O₂ (100 µM) + HK (20 µM) and H₂O₂ (100 µM) + NAC (1 mM) on (A) ROS production and (B) lipid peroxidation of MC3T3-E1 cells after 72 h. *p < 0.05, **p < 0.01, ***p < 0.001, relative to control group.

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Oxidative stress could stem from deregulation of the intracellular oxidation–antioxidation balance and is linked with a significant increase in intracellular ROS levels. Upregulation of ROS could lead to biomolecule oxidation such as lipids, proteins, and DNA, which would result in tissue damage (Juan et al., 2021). Lipid peroxides (MDA) can lead to elevated cell and mitochondrial membrane damage and associated apoptosis (Su et al., 2019). It has been documented that the overproduction of ROS contributes to the deregulation of mineral tissue homeostasis and causes bone remodeling mediated by decreasing bone formation and upgrading bone resorption (Marcucci et al., 2023). In the present study, although H₂O₂ increased the production of ROS and MDA, after co-treatment with HNK for 72 h, these effects were reversed to a significant extent.