Home Physical Sciences Protective activities of silver nanoparticles containing Panax japonicus on apoptotic, inflammatory, and oxidative alterations in isoproterenol-induced cardiotoxicity
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Protective activities of silver nanoparticles containing Panax japonicus on apoptotic, inflammatory, and oxidative alterations in isoproterenol-induced cardiotoxicity

  • Xiao Xu , Zhipeng Diao , Bo Zhao , Huajuan Xu , Shuying Yan and Huilin Chen EMAIL logo
Published/Copyright: April 3, 2024

Abstract

Panax japonicus has long been utilized as an herbal remedy in Chinese traditional medicine for treating various diseases. In this investigation, we present the environmentally friendly silver nanoparticle (AgNP) synthesis by the aqueous extract of P. japonicas to follow its cardioprotective effects. Through various analytical methods, we identified the nanoparticles (NPs). Our XRD findings revealed the formation of Ag@P. japonicus, while FE-SEM imagery indicated a spherical shape, with NPs measuring less than 40 nm. The UV–Vis and FT-IR spectroscopy confirm the green synthesis of Ag@P. japonicus. In the medicinal section, 45 Wistar rats were utilized. These groups consisted of a normal group, a group that was solely treated with isoproterenol for inducing myocardial infarction, and two groups that were pretreated with AgNPs at different doses for 14 days. These pretreated groups were then challenged with isoproterenol. The expression of PI3K/Akt/mTOR and other downstream inflammatory and apoptotic mediators were followed. Additionally, the expression of Keap1, Nrf2, ECG, cardiac markers, and other downstream antioxidant enzymes were assessed. Treatment with AgNPs ameliorated the apoptosis, inflammation, and myocardial autophagy, regulated the PI3K/Akt/mTOR pathway, increased the antioxidant enzyme efficacies, and activated the Keap1/Nrf2/HO-1 pathway. The findings suggest that AgNPs may have a cardioprotective efficacy on myocardial infarction by mitigating the Keap1/Nrf2 pathway, GST, GPx, GSH, SOD, IL-1β, IL-6, TNF-α, NF-κB, Bax, Bcl2, caspase-9, caspase-3, and PI3K/Akt/mTOR pathway. Furthermore, the treatment decreased the infarct region size, attenuated the cardiac indicators levels, and mitigated immune cell infiltration and myocardial necrosis.

1 Introduction

The Panax genus (Araliaceae family) is comprised of roughly 20 species. These plants have gained significant recognition for their extensive application as food, functional food, dietary supplements, and medicinal materials in Chinese Traditional Medicine over thousands of years. Four notable plants within the Panax genus are Panax notoginseng, Panax quinquefolius, Panax vietnamensis, and Panax japonicus [1]. It is primarily found in the wild in Korea, Japan, and China. It is also referred to as “Zhujieshen” and serves as a significant alternative to ginseng roots among minority ethnic groups [2,3]. In Chinese Traditional Medicine, this plant is utilized as an agent for treating various conditions such as bleeding, meridian blockage, pain relief, enhanced circulation, and spleen and stomach invigoration [4]. Modern research indicates that P. japonicus has a crucial role in managing several diseases, including rheumatic arthritis, cancer, and cardiovascular ailments [5]. Studies have revealed that the key active components in P. japonicus are saponins, polysaccharides, phenolic acids, and alkaloids [6]. Among these, ginsenosides, a type of triterpenoid saponins, are predominant in P. japonicus. They exhibit numerous pharmacological effects that play a critical role in treating various cancers, cardiovascular issues, neuronal disorders, inflammation, immune regulation, and metabolic disorders [5]. In particular, Ginsenoside Re, which is abundant in P. japonicus, demonstrates the ability to inhibit cardiomyocyte apoptosis [7].

Among the several green-synthesized nanoparticles (NPs), silver nanoparticles (AgNPs) have emerged as highly valuable nanomaterials for numerous industrial applications and have already been successfully commercialized. Recently, there has been a growing focus on bioinspired AgNPs due to their increasing demand and the green synthesis significant advantages. AgNPs have found extensive usage in biomedical, medical, and textile industries, sunscreen, catalysis, food preservation, and cosmetics [8,9]. Numerous studies have explored the formation of AgNPs using aqueous herbal plant extracts. These extracts contain various secondary metabolites, including steroids, terpenoids, glycosides, flavonoids, saponins, tannins, phenolics, and alkaloids [10,11]. Among these phytochemical compounds, saponins have acquired considerable attention in recent times due to their potential pharmaceutical and amphiphilic nature usages. Saponins consist of two sections: hydrophobic triterpenoid covalently attached hydrophilic sugar units or steroid components [12]. In environmental applications, the saponins have been utilized as biosurfactants [13]. In the AgNP synthesis context, it has been discovered that saponins can serve as effective surfactants for capping NPs. The capping agents have a critical role in the AgNP synthesis and can significantly influence the efficacy of the synthesis process [10,14]

In this investigation, we present the environmentally friendly AgNP synthesis by the P. japonicas aqueous extract. The NPs were characterized by UV–Vis and FT-IR spectroscopy, FE-SEM and TEM imaging, XRD, and EDX analysis. The AgNPs were examined for their protective activities on immunological, cellular, and molecular changes in isoproterenol-induced cardiotoxicity. Furthermore, the signaling of Keap1/Nrf2 pathway, GST, GPx, GSH, SOD, IL-1β, IL-6, TNF-α, NF-κB, Bax, Bcl2, caspase-9, and caspase-3 and PI3K/Akt/mTOR pathway were evaluated.

2 Materials and methods

2.1 Chemical and instruments

Silver nitrate and isoproterenol were purchased from Merck company, Germany. ELIZA kit was purchased from the Pars Azmoon company, Iran.

A Spekol 2000 was used for the recording of UV–Vis spectra; the FT-IR spectrum was recorded using a KBr disc at the range of 400–4,000 cm−1 using a Shimadzu FT-IR 8400; MIRA3TESCAN-XMU was used to report the FE-SEM images and EDS result. A Hitachi Transmission Electron Microscopes model 7800 was used to record TEM images. The XRD pattern was recorded in the 2θ range of 20–80° by an STOE instrument at a voltage of 40 kV, a current of 30 mA, and Cu-Kα radiation.

2.2 Synthesis of the AgNPs (Ag@P. japonicus)

A 10 g of dried leaves of P. japonicus was boiled in 20 mL of deionized water for 10 min. The extract was filtered after cooling. To synthesize Ag@P. japonicus, 20 mL of the extract was poured into 50 mL of AgNO3 (0.1 M). The reaction mixture was stirred for 12 h at 50°C. The clear solution was converted to a black cloudy one during the reaction. After that time, the AgNPs were separated using the centrifuging method at 15,000 RPM for 12 min. The NPs finally were put at 45°C to dry in an oven.

2.3 Following the cardioprotective efficacies of Ag@P. japonicus

In the recent study, a total of 40 Wistar rats were utilized to follow the protective efficacies of AgNPs containing P. japonicus on isoproterenol-induced cardiotoxicity. The study focused on the Keap1/Nrf2/HO-1 and PI3K/Akt/mTOR pathways. The rats were divided into four groups: a normal group, a group of myocardial infarction induced with isoproterenol, and two groups pretreated with different doses of AgNPs containing P. japonicus (500 or 100 µg/kg).

The pretreatment lasted for 14 days, and isoproterenol was administered on the 13th and 14th days. The rats were anesthetized by urethane at the end of the experiment and placed in a non-invasive computerized ECG apparatus to record ECG readings.

The blood samples were centrifuged to separate their serum for biochemical assays. The hearts of the rats were dissected, weighed, and stored at −80°C. The heart’s infarction size was determined by studying the histopathological slides and using ImageJ® software.

NF-kB, TNF-α, IL-6, IL-1, caspase-9, and caspase-3 were measured in the extracted heart tissues using ELISA kits. Western blotting examination was conducted to measure the mTOR, Akt, PI3K, HO-1, NRf2, and Keap 1 protein expression. Additionally, a real-time PCR test was used to determine the gene expression levels of mTOR, Akt, PI3K, HO-1, NRf2, and Keap 1.

Antioxidant markers and the levels of Ca2+, mitochondrial ATP, and GSH were determined in the prepared mitochondria by assay kits following the manufacturer’s protocols.

2.4 Statistical analysis

To perform statistical calculations, SPSS version 18 software (one-way ANOVA statistical test) was used to compare different groups and Duncan’s support test. The limit of statistical inference was considered p < 0.05.

3 Results and discussion

3.1 Chemical characterization of Ag@P. japonicus

The SPR of Ag@P. japonicus was analyzed by UV–visible spectroscopy. Figure 1 reveals the Ag@P. japonicus spectrum. A band at 223,261, 337, and 457 nm indicates the silver ions reduction by P. japonicus extract and the formation of Ag@P. japonicus. Similar peaks have been reported for AgNPs by other researchers [15,16]

Figure 1 
                  Surface plasmon resonance spectrum of AgNPs prepared using P. japonicus extract.
Figure 1

Surface plasmon resonance spectrum of AgNPs prepared using P. japonicus extract.

The qualitative analysis of FT-IR spectroscopy was applied to approve the green synthesis of Ag@P. japonicus. In this technique, the appearance of the peaks at wavenumbers up to 700 cm−1 is assigned for metal bonds. For Ag@P. japonicus, the peaks can be observed at 470 and 513 cm−1 (see Figure 2). Similar peaks have been reported for AgNPs synthesized using Salvia leriifolia [17]. In addition, the peaks for the secondary metabolites of P. japonicus extract can be discerned at 1,026, 1,534–1,724, 2,920, and 3,292 cm−1 for functional groups of C–O, C═C, C═O, C–H, and O–H that found in secondary metabolites of P. japonicus.

Figure 2 
                  The FT-IR spectrum of AgNPs prepared using P. japonicus extract (KBr disc, in the region from 400 to 4,000 cm−1).
Figure 2

The FT-IR spectrum of AgNPs prepared using P. japonicus extract (KBr disc, in the region from 400 to 4,000 cm−1).

The results for the qualitative analysis of EDS and elemental mapping of Ag@P. japonicus are presented in Figures 3a and b, respectively. The present signals in the EDS diagram (Figure 3a) include signals around 0.3 and 0.5 keV for C Lα and O Lα, respectively. Two signals of less and more than 3.0 keV, which are respected to Ag Lα and Ag Lβ, approve the formation of Ag@P. japonicus. Dakshayani et al. have reported a similar signal for the elements of AgNPs synthesized using Selaginella extract [18]. Furthermore, quantitative analysis revealed the percentage of 11.03, 25.55, and 63.42% for silver, oxygen, and carbon. The two last elements are associated with the organic compounds of P. japonicus that are linked to the synthetic NPs’ surface.

Figure 3 
                  EDX diagram of AgNPs prepared using P. japonicus extract.
Figure 3

EDX diagram of AgNPs prepared using P. japonicus extract.

The crystallinity of Ag@P. japonicus was examined by the XRD technique, and according to the finding from the XRD graph of Ag@P. japonicus (Figure 4), the AgNPs’ crystallinity was confirmed. The signals at 37.77 (111), 43.56 (200), 64.12 (220), and 76.95 (311) are well matched to data of the standard JCPD card 04-0783 and previous reports for green-synthesized AgNPs [19,20]. The Debye Scherrer equation gave a crystal size of 28.88 nm for Ag@P. japonicus

Figure 4 
                  The XRD diagram of AgNPs prepared using P. japonicus extract. The graph was recorded in the 2θ range of 20–90° at a voltage of 40 kV, a current of 30 mA, and Cu-Kα radiation.
Figure 4

The XRD diagram of AgNPs prepared using P. japonicus extract. The graph was recorded in the 2θ range of 20–90° at a voltage of 40 kV, a current of 30 mA, and Cu-Kα radiation.

The morphology of Ag@P. japonicus was investigated using TEM and FE-SEM (see Figure 5). The analysis shows the successful formation of the AgNPs in a spherical structure with an estimated size of less than 40 nm. The aggregation of Ag@P. japonicus has emerged in the FE-SEM image as well as TEM one, which is well-known as a familiar character for metallic NPs in literature [15,17,21,22]. The reported size for green synthetic AgNPs using different plants ranges from 5 to less than 80 nm [1420,23].

Figure 5 
                  (a) TEM image of AgNPs prepared using P. japonicus extract; (b) FE-SEM image of AgNPs prepared using P. japonicus extract.
Figure 5

(a) TEM image of AgNPs prepared using P. japonicus extract; (b) FE-SEM image of AgNPs prepared using P. japonicus extract.

3.2 Cardioprotective efficacies of Ag@P. japonicus

In this investigation, isoproterenol was utilized to induce cardiotoxicity. Isoproterenol-induced cardiotoxicity serves as a well-established model for examining the myocardial ischemia pathophysiology. Isoproterenol stimulates contractility and heart rate, thereby raising the consumption of myocardial oxygen [24,25]. The extra elevation in heart stimulation can potentially result in ischemia and subsequently lead to cardiotoxicity. During infarction and ischemia, myocytes result in reduced ATP production, which can compromise membrane integrity and lead to myocardial dysfunction and calcium overload [26]. Following an ischemic insult, the sudden restoration of oxygen supply yields ROS, particularly by mitochondria, which more damages myocytes [2730]. Moreover, there is substantial evidence supporting the reduction in the PGC-1α and mitochondrial transcription factor A expression in various animal models of heart failure, accompanied by mitochondrial dysfunction and oxidative stress [30]. Also, NF-κB is related to the NLRs superfamily which is expressed highly in cardiac dysfunction. Therefore, targeting these genes for the treatment of cardiotoxicity holds significant promise [31].

The ECG traces of both the normal group and the Ag@P. japonicus group in this study were found to be normal. However, the group with isoproterenol-induced myocardial infarction exhibited several ECG alterations, such as R–R and P–R intervals, QT interval, QRS complex, a shorter P wave, and a wider ST segment compared to the control group. Interestingly, pretreatment with Ag@P. japonicus at doses 50 or 100 µg/kg reversed most of these ECG alterations, as shown in Figure 6.

Figure 6 
                  Effects of AgNPs prepared using P. japonicus extract on ECG components (ST elevation (mV), QT interval (s P wave (s), QRS complex (s), P–R interval (s), and R–R interval (s)).
Figure 6

Effects of AgNPs prepared using P. japonicus extract on ECG components (ST elevation (mV), QT interval (s P wave (s), QRS complex (s), P–R interval (s), and R–R interval (s)).

Animals treated with isoproterenol exhibited a notable increase in heart-to-body ratios and the infarct areas presence. Conversely, the control group and the Ag@P. japonicus group displayed normal heart-to-body ratios and minimal infarct areas. Furthermore, the Ag@P. japonicus group demonstrated a decrease in the creatine kinase-myocardial bound, creatine phosphokinase, cardiac troponinin I, and cardiac troponin T levels when compared to the untreated group (Figure 7).

Figure 7 
                  Effects of AgNPs prepared using P. japonicus extract on creatine kinase-myocardial bound (IU/L), creatine phosphokinase (IU/L), infarct size (%), cardiac troponin T (ng/mL), cardiac troponin I (ng/mL), and heart/weight (%).
Figure 7

Effects of AgNPs prepared using P. japonicus extract on creatine kinase-myocardial bound (IU/L), creatine phosphokinase (IU/L), infarct size (%), cardiac troponin T (ng/mL), cardiac troponin I (ng/mL), and heart/weight (%).

To evaluate the impact of Ag@P. japonicus on the mitochondrial antioxidant status, we measured the antioxidant enzymes GST, GPx, CAT, and SOD activities and the non-enzymatic antioxidant GSH levels in several treatment groups (Figure 8). The results showed a significant decrease in both GSH levels and the mitochondrial antioxidant enzyme activities in animals challenged with isoproterenol, compared to the control group. However, in the groups treated with Ag@P. japonicus, the levels of these enzymes were found to increase.

Figure 8 
                  Effects of AgNPs prepared using P. japonicus extract on GST (nmol/mg protein), SOD (micromole H2O2/min/mg protein), CAT (micromole H2O2/min/mg protein), GPx (µg of GSH utilized/min/mg protein), and GSH (nmol/mg protein).
Figure 8

Effects of AgNPs prepared using P. japonicus extract on GST (nmol/mg protein), SOD (micromole H2O2/min/mg protein), CAT (micromole H2O2/min/mg protein), GPx (µg of GSH utilized/min/mg protein), and GSH (nmol/mg protein).

The protein and mRNA expression levels of mTOR, Akt, PI3K, HO-1, NRf2, and Keap 1 were altered by the challenge with isoproterenol. However, the pretreatment with AgNPs resulted in a notable reduction in the mRNA and protein expression levels of HO-1, NRf2, and Keap 1, while simultaneously increasing the mRNA and protein expression levels of mTOR, Akt, and PI3K (Figure 9).

Figure 9 
                  Effects of AgNPs prepared using P. japonicus extract on Keap 1, NRf2, HO-1, PI3K, Akt, and mTOR mRNA (A) and protein (B) levels.
Figure 9

Effects of AgNPs prepared using P. japonicus extract on Keap 1, NRf2, HO-1, PI3K, Akt, and mTOR mRNA (A) and protein (B) levels.

The levels of inflammatory markers were found to be similar in both the control group and the Ag@P. japonicus group. However, the markers showed a significant increase in response to isoproterenol-induced cardiotoxicity (Figure 10).

Figure 10 
                  Effects of AgNPs prepared using P. japonicus extract on anti-inflammatory cytokines (IL-1β (pg/mg protein), IL-6 (pg/mL), TNF-α (pg/mg protein), and NF-kB (pg/mg protein)) levels.
Figure 10

Effects of AgNPs prepared using P. japonicus extract on anti-inflammatory cytokines (IL-1β (pg/mg protein), IL-6 (pg/mL), TNF-α (pg/mg protein), and NF-kB (pg/mg protein)) levels.

Following Figure 11, there was no disparity observed in the protein levels (caspase-3 and caspase-9 (ng/mL)) and mRNA expression (Bcl2 and Bax) between the Ag@P. japonicus group and the control group. However, the markers exhibited a notable increase in response to isoproterenol-induced cardiotoxicity.

Figure 11 
                  Effects of AgNPs prepared using P. japonicus extract on levels of protein (caspase-9 and caspase-3 (ng/mL)) and mRNA (Bcl2 and Bax) expression.
Figure 11

Effects of AgNPs prepared using P. japonicus extract on levels of protein (caspase-9 and caspase-3 (ng/mL)) and mRNA (Bcl2 and Bax) expression.

In the cohort of animals with isoproterenol-induced cardiotoxicity, there was a notable increase in the levels of Ca2+ in the mitochondria of cardiac cells. Conversely, the levels of mitochondrial ATP were reduced. However, when the animals were pretreated with Ag@P. japonicus, the mitochondrial Ca2+ and ATP content levels were improved in comparison to the group treated with isoproterenol (Figure 12).

Figure 12 
                  Effects of AgNPs prepared using P. japonicus extract on mitochondrial ATP (nM/mg protein) and Ca2+ (nM/mg protein).
Figure 12

Effects of AgNPs prepared using P. japonicus extract on mitochondrial ATP (nM/mg protein) and Ca2+ (nM/mg protein).

AgNPs are extensively utilized as nanocarriers for delivering cardioprotective drugs. The AgNP utilization in combination with recent clinical drugs has been proposed as a new approach with raised efficacy for heart disease treatment. Ag nanocarriers are favored because of their low immunogenicity, low toxicity, synthesis ease, and stability [32]. Studies have shown that clinical drugs exhibit increased efficiency and accuracy when conjugated with NPs. Simdax, a confirmed drug for heart disease treatment, when combined with NPs, demonstrates cardioprotective efficacies in doxorubicin-induced heart failure rats. The nanoconjugates and simdax cardioprotective efficacies surpass those of their counterparts because of the nanoconjugates’ enhanced targeting of the injured tissue [33]. Metoprolol is combined with NPs to improve delivery to cardiac tissues [34]. miR155-NPs can be used to treat diabetic cardiomyopathy [34,35]. The in vivo miR155-NPs administration conjugates led to a significant rise in anti-inflammatory type 2 macrophages and a reduction in inflammation. Consequently, this led to a decrease in cell apoptosis and ultimately contributed to the cardiac function restoration [35].

Also, the cardioprotective efficacies of other metallic NPs have been indicated in previous studies [3640]. Metal and metal oxide NPs possess distinct properties that can potentially impact the management and treatment of cardiovascular diseases [37,38]. The utilization of gold NPs in genetic technologies has been sanctioned by the FDA. These technologies are presently being explored through the identification of numerous molecular and genetic biomarkers [38,39]. Furthermore, additional types of NPs, like superparamagnetic iron oxide NPs, are recognized as secure and effective MRI contrast agents for assessing therapy or focusing on specific molecules. It is worth mentioning that precise management of the physicochemical properties of NPs greatly expedites the advancement of personalized medical treatments for individual patients [3640]. NPs have shown significant progress in the treatment of cancer and hereditary diseases, whereas their application in managing or treating cardiovascular diseases is still in its early stages. However, they have demonstrated great potential for various uses [3640], as illustrated in Table 1.

Table 1

Cardioprotective efficacies of several NPs with mentioning their therapeutic dose, remedial agent, and size in different models of animals

Nanoparticles Results Dose Animal model Remedial agent Size (nm)
Graphene oxide gold nanosheets [36] The cardiac contractility has been enhanced, leading to the restoration of ventricular functions. 5 × 2 mm scaffold Rat Chitosan-graphene oxide gold nanosheets scaffold <100
Graphene NPs [37] The size of the infarct was decreased, leading to enhancements in both capillary density and cardiac performance. 300 µL, intramyocardial Rat VEGF <40
Gold NPs [38] Myocardial injury was ameliorated (400 µg/kg/day) intravenous Rat Au 50
Gold NPs [39] Reduced infarction size, enhanced systolic performance, suppressed cardiac fibrosis, no impact on apoptosis or hypertrophy. 100 µL/day intravenous Mouse PEG coated 10
Copper NPs [40] Reduced infarct size, diminished levels of oxidative stress, inflammatory cytokines, and apoptosis were observed. 1 mg/kg/day, p.o Rat Cu <100

4 Conclusion

To create the AgNPs, we followed a green synthesis approach utilizing the P. japonicus aqueous extract. The resulting Ag@P. japonicus was characterized using a variety of analytical examinations, including FE-SEM, FT-IR, UV–Vis, EDX, and XRD. The FT-IR spectrum confirmed the formation of Ag@P. japonicus, while XRD analysis revealed the AgNPs’ crystalline nature. TEM and FE-SEM imaging indicated a spherical structure for the synthesized NPs, with an observed tendency towards aggregation, a common property among metallic NPs. The AgNPs have shown positive effects on various cardiac markers, such as ECG, Nrf2, and Keap1 expression. Additionally, they have influenced the expression of PI3K/Akt/mTOR and other downstream inflammatory and apoptotic mediators. The NP treatment decreased the infarct region size, attenuated the cardiac indicators levels, and mitigated immune cell infiltration and myocardial necrosis. These NPs hold great potential for numerous medical usage in the pharmaceutical industry, particularly in the new formulation development to combat cardiotoxicity. All, the present research revealed that green synthetic AgNPs provide valuable insights into the characteristics and potential applications to serve as cardioprotective and antioxidant agents. However, more investigations on AgNPs especially in vivo studies are required to introduce the AgNP as a drug for oxidative alterations in isoproterenol-induced cardiotoxicity.

  1. Funding information: This work was supported by the Medical Research Project of Hongkou District Health and Wellness Committee (Grant fund number: 1902-32).

  2. Author contributions: Xiao Xu: Conceptualization, data curation, formal analysis, acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, and writing – review and editing. Zhipeng Diao: conceptualization, data curation, formal analysis, resources, software, supervision, validation, visualization, writing – original draft. Bo Zhao: project administration, resources, software, supervision, validation, visualization, writing – original draft, and writing – review and editing. Huajuan Xu: conceptualization, data curation, formal analysis, acquisition, investigation, methodology, project administration, resources, software, supervision. Shuying Yan: conceptualization, data curation, formal analysis, acquisition, investigation, methodology, project administration. Huilin Chen: conceptualization, data curation, formal analysis, acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, and writing – review and editing.

  3. Conflict of interest: There is no any conflict of interest.

  4. Ethical approval: The experiments were performed according to the ethical guidelines of the International Association for the Study of Humans.

  5. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Received: 2024-01-25
Revised: 2024-03-06
Accepted: 2024-03-08
Published Online: 2024-04-03

© 2024 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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