Xinshuaining preparation protects H9c2 cells from H₂O₂-induced oxidative damage through the PI3K/Akt/Nrf-2 signaling pathway

ABSTRACT

Background

Cardiovascular disease (CVD) is the leading cause of death. Oxidative stress is an important pathological process of a variety of CVDs. Xinshuaining preparation has a therapeutic effect on the heart failure. However, the anti-oxidative stress role of Xinshuaining preparation in H9c2 cells is still unclear.

Methods

The medicated serum of Xinshuaining preparation was acquired and utilized to hatch with H₂O₂-induced H9c2 cells. Main components in the Xinshuaining preparation were analyzed by liquid chromatography-mass spectrometry (LC/MS). The effect of medicated serum on the cell viability, apoptosis rate, the oxidative stress indicators (SOD, GSH-Px, and MDA), mitochondrial membrane potential (MMP), and ROS level was evaluated by CCK-8, flow cytometry, commercial biochemical detection kits, and JC-1 staining. Additionally, the associated mechanism was determined by the detection of the protein levels (PI3K, phosphorylated PI3K, Akt, phosphorylated Akt, and Nrf-2) through western blot assays, which was also further assessed with the application of LY294002.

Results

The medicated serum of Xinshuaining preparation notably increased the H₂O₂-reduced, the cell viability, the concentration of SOD and GSH-Px, MMP level and the relative protein expression level of phosphorylated PI3K and Akt and Nrf-2, while dampened the H₂O₂-elevated the level of the cell apoptosis rate, MDA, and ROS. However, Xinshuaining preparation on the cell viability, apoptosis, and oxidative stress was notably antagonized by LY294002 pre-treatment.

Conclusions

The medicated serum of Xinshuaining preparation increased the cell viability and suppressed apoptosis and oxidative stress via the PI3K/Akt/Nrf-2 signaling pathway.

KEYWORDS:

• Cardiovascular disease
• Xinshuaining preparation
• oxidative stress
• PI3K/Akt pathway
• Nrf-2

Introduction

Cardiovascular disease (CVD) is the leading cause of death and disability that affects approximately one third people around the world (https://www.idf.org/our-activities/care-prevention/cardiovascular-disease.html). The World Health Organization estimates that CVD results in 17.9 million deaths in 2019 and accounted for one-third of all deaths worldwide. Although a huge number of efforts have been devoted into the CVD studies, its clinic outcome is still dull. Moreover, the growing cost on CVD has caused enormous burden to patients’ families. Therefore, it is extremely important and indispensable to deeply explore the underlying mechanisms and seek potential therapeutic targets and drugs for CVD.

As one of the crucial mechanisms involved in CVD, oxidative stress is a state of imbalance between oxidation and anti-oxidation in the body, as indicated by the excessive production of reactive oxygen species (ROS), the degree of oxidation exceeding the scavenging ability of antioxidants, and the imbalance of the oxidative system and the antioxidant system (1). Plenty of studies have shown that inhibition of oxidative stress can effectively protect heart against injury, and thus, oxidative stress has been one of the therapeutic targets in CVD (2).

Xinshuaining preparation is mainly developed from traditional Chinese medicines (TCM) including Codonopsis pilosula (Franch.) Nannf (3), Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (4), Atractylodes macrocephala Koidz (5), Poria cocos (Schw.) Wolf (6), Cinnamomum cassia Presl (7), Salvia miltioγrhiza Bge (8), Descurainia sophia (L.) Webb. ex Prantl (9), Clematis chinensis Osbeck (10), and Aconitum carnichaelii Debx (11), which have shown cardioprotective effects. Furthermore, previous studies have reported that the Xinshuaining mixture combined with conventional western medicine has a better curative effect than western medicine alone in the treatment of patients with coronary heart disease ischemic cardiomyopathy type heart failure (12,13). However, the anti-oxidation role of Xinshuaining preparation in the heart still needs more attention.

Thus, in the present study, the effect and the underlying mechanisms of Xinshuaining preparation on the H9c2 cells induced by H₂O₂ were investigated. The results showed that Xinshuaining preparation protected H9c2 cells from H₂O₂-induced oxidative damage through the PI3K/Akt/Nrf-2 signaling pathway.

Materials and methods

Liquid chromatography-mass spectrometry (LC/MS) analysis

Xinshuaining decoction was centrifuged at 12000 r/min for 10 min to collect the supernatants. After being filtered via the 0.45 µm membrane, 10 µL of supernatant samples were prepared for following examination. The High-Performance Liquid Chromatography (HPLC) system LC-30 (SHIMADZU, Kyoto, Japan) was connected with a mass spectrometer SCIEX 5600 (AB Sciex Instruments, United States). The column temperature was 35°C. Mobile phase A was acetonitrile, and mobile phase B was 0.1% HCOOH-H₂O. The chromatographic separation was conducted with a SHIMADZU InerSustain C18 (100 × 2.1 mm, 2 µm). Modes with electrospray ionization (ESI) positive ion and negative ion were used in the current study severally. The ESI source conditions are listed as follows: Ion Source Gas1 (Gas 1): 50; Gas 2: 50; Curtain Gas (CUR): 25; Source Temperature: 500°C (positive ion) and 450°C (negative ion); Ion Sapary Voltage Floating (ISVF) 5500 V (positive ion) and 4400 V (negative ion); TOF MS scan range: 100–1200 Da; product ion scan range: 50–1000 Da; TOF MS scan accumulation time 0.2s; and product ion scan accumulation time 0.01s. The secondary mass spectrum was acquired through information-dependent acquisition (IDA) and adopts high sensitivity mode: declustering potential (DP): ±60 V and collision energy: 35 ± 15 eV.

Animal

Adult Sprague-Dawley rats (age: 7–8 weeks, weight: 200–220 g) were bought and acclimated to standard laboratory conditions for 7 days before experiments. Rats were fed with standard diet and water ad libitum at (25 ± 2) °C and a relative humidity of 40%-60% with a 12-hour/12-h light-dark cycle. All the procedures were performed severely according to the National Institute of Health Guide for the Care and Use of Laboratory Animals. This study was also authorized by the Board and Ethics Committee of Affiliated Hospital of Chengdu University of Traditional Chinese Medicine (Approval number: 20220613004).

Preparation of medicated serum

Twenty rats were divided into two groups in random (n = 10), including control and Xinshuaining preparation groups. Rats in Xinshuaining preparation group were intragastrically treated with 31.2 g/(kg.d) Xinshuaining decoction, whereas rats in the control group were intragastrically administered with the same amount of saline a day for seven consecutive days. Rats were intraperitoneally anesthetized with sodium pentobarbital (40 mg/kg), and then blood was taken from the abdominal aorta. Serum was separated and inactivated the complement for further in vitro experiments.

Cell culture

H9c2 cardiomyocyte cells were bought from Procell (CL-0089, Wuhan, China) and were maintained in high glucose DMEM (Solarbio, Beijing, China) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/ streptomycin (Solarbio) at 37°C and 5% carbon dioxide (CO₂). On the one hand, cells were incubated with different concentrations of medicated serum (0, 5, 10, 15 and 20%) or different concentrations of H₂O₂ (0, 10, 25, 50, 75, 100, and 150 μM) for 48 h. On the other hand, cells were divided into six groups, including control, H₂O₂, H₂O₂+ Negative serum, H₂O₂+ Medicated serum, H₂O₂+ LY294002, and H₂O₂+ Medicated serum+LY294002. Cells in the H₂O₂ group were treated with 75 μM H₂O₂, cells in the H₂O₂+ Negative serum group were treated with 75 μM H₂O₂ and serum from rats injected with saline, cells in the H₂O₂+ Medicated serum group were treated with 75 μM H₂O₂ and 10% medicated serum, cells in the H₂O₂+ LY294002 group were treated with 75 μM H₂O₂ and 25 μM LY294002, cells in the H₂O₂+ Medicated serum+LY294002 group were treated with 75 μM H₂O₂ and 10% medicated serum as well as 25 μM LY294002, and cells in the control group were treated with the same amount of phosphate buffer saline (PBS, Solarbio).

Cell Counting Kit-8 Assay

H9c2 cells were seeded in 96-well plates with an inoculation density of 1 × 10⁵/well and maintained for 24 h with 5% CO₂ at 37°C. After cells were suffered from different treatments, the Cell Count Kit-8 (Dojindo Laboratories, Kumamoto, Japan) was utilized to examine the proliferation of cells based on the operating instruction. The absorbance was detected at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Flow cytometry

The apoptosis of H9c2 cells was assessed by the flow cytometry assay. Cells were gathered, resuspended with 500 μL of binding Buffer, and then stained with a mixture of 5 μL of phycoerythrin (PE) (Sigma-Aldrich) and 5 μL of allophycocyanin (APC) (Sigma-Aldrich) at room temperature for 15 min in the dark. In addition, the level of ROS was detected using a Reactive oxygen species assay kit (S0033, Beyotime, Shanghai, China) according to the operating instruction. The apoptosis rate and the fluorescence of H9c2 cells were analyzed by flow cytometry (BD FACSVerse, USA).

Biochemical detection

The concentration of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) was measured using a commercial Total superoxide dismutase (T-SOD) test kit (A001-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), a Malondialdehyde (MDA) test kit (A003-1-1, Nanjing Jiancheng Bioengineering Institute), and a Glutathione Peroxidase (GSH-Px) test kit (A005-1-2, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s protocol. The absorbance of wells was detected at 532 nm (MDA), 560 nm (SOD), and 412 nm (GSH-Px) with a microplate reader (Thermo Fisher Scientific).

JC-1 staining

H9c2 cells were cultured on coverslips and incubated with 0.1 μM JC-1 (Molecular Probes) for 20 min at 37°C. CCCP (50 mM) acted as a positive control and hatched for 20 min before JC-1 staining. Fluorescence images were acquired using a laser-scanning confocal microscope (Zeiss LSM 710 META, Germany) with an excitation at 525 nm and 490 nm, respectively. Changes in mitochondrial membrane potential (MMP) were evaluated by the ratio of aggregated JC-1 (525 nm, red fluorescence) to monomeric JC-1 (490 nm, green fluorescence).

Western blot assay

Nuclear and cytoplasmic proteins of H9c2 cells were isolated using a ExKine Nuclear and Cytoplasmic Protein Extraction Kit (KTP3001, Abbkine, California, USA), and the concentration of protein as sample was measured using a Protein Assay kit (Beyotime, Shanghai, China). Then, protein samples were resolved by 10% SDS-PAGE gel and electrically transferred to PVDF membranes (Millipore, MA, USA). Next, the membranes were hatched with the primary antibodies (PI3K, 1:1000, ab131067; phosphorylated PI3K, 1:1000, ab278545; Akt, 1:500, ab8805; phosphorylated Akt, 1:1000, ab38449; Nrf-2, 1:2000, ab137550; β-actin, 1: 5000, ab8227; Lamin A, 1:1000, ab8980; Abcam, Cambridge, UK) overnight at 4°C after being blocked with 3% bovine serum albumin (BSA) at room temperature for 1 h. Subsequently, the membranes were rinsed with TBST three times and then hatched with goat-anti-rabbit IgG (H + L)-HRP (1:10000, ab6721, Abcam) at room temperature for 1 h. Protein bands were visualized using an Electrochemiluminescence (ECL) chemiluminescence kit (WBULS0500; EMD Millipore), and the bands intensity was analyzed using Image-Pro Plus 6.0 software.

Statistical analysis

Data were exhibited as the means ± SD. Data among multiple groups were analyzed by ANOVA, among which the LSD-t test was applied for groups with homogeneous variance, and Tamhane’s T2 test was used for groups with unequal variance using the SPSS 22.0 statistical software (IBM, Armonk, New York, USA) followed by the Post Hoc Bonferroni test. The differences were thought as statistically significant when p < .05.

Results

The analysis of active ingredients of Xinshuaining decoction

Results in Figure 1 display the chromatograms of positive (Figure 1(a)) and negative (Figure 1(b)) ions of Xinshuaining decoction. For preprocessing, containing peak extraction, noise removal, deconvolution, and peak alignment, the raw data of LC-MS were put into MS-DIAL 4.60 software. The extracted peak information was compared with GNPS, Respect, and MassBank databases. The detailed active ingredients of the chromatograms of positive and negative ion of Xinshuaining decoction are listed in Table S1.

Figure 1. The chromatograms of positive and negative ions of the Xinshuaining decoction sample. (A) The chromatograms of positive ions of the Xinshuaining decoction sample. (B) The chromatograms of negative ions of the Xinshuaining decoction sample.

The screen of medicated serum of Xinshuaining preparation and H₂O₂ concentration

The concentrations of medicated serum and H₂O₂ were first screened after H9c2 cells were treated with 0, 5%, 10%, 15%, and 20% medicated serum or 0, 10, 25, 50, 75, 100, and 150 μM H₂O₂. CCK-8 results showed that 20% medicated serum observably reduced the viability of H9c2 cells, while 15% medicated serum just decreased the cell viability with no statistical difference compared with those treated with 0, 5%, and 10% medicated serum (Figure 2(a)). No statistical difference was observed in the viability of H9c2 cells between 15% and 20% medicated serum treatment (Figure 2(a)). Thus, 10% medicated serum was chosen for subsequent experiments. In addition, the viability of H9c2 cells was notably diminished with all the 75, 100, and 150 μM H₂O₂ treatment, and no statistical difference was shown among these three groups (Figure 2(b)). Therefore, 75 μM H₂O₂ was used for further assays. Taken together, 10% medicated serum and 75 μM H₂O₂ were utilized for subsequent assays.

Figure 2. 10% medicated serum and 75 μM H₂O₂ were utilized for subsequent assays. H9c2 cells were treated with 0, 5%, 10%, 15%, and 20% medicated serum or 0, 10, 25, 50, 75, 100, and 150 μM H₂O₂, and then the cell viability was detected by CCK-8 assay. (A) The cell viability of H9c2 cells was determined by CCK-8 after being treated with different concentrations of medicated serum. (B) The cell viability of H9c2 cells was examined by CCK-8 after being treated with different concentrations of H₂O₂. The means ± SD of four independent samples are shown. *p < .05.

The medicated serum of Xinshuaining preparation increased the cell viability and inhibited apoptosis and oxidative stress in H9c2 cells

Then, the effect of medicated serum of Xinshuaining preparation on the H9c2 cells was assessed. The results revealed that medicated serum not negative serum prominently enhanced the H₂O₂-induced reduction of cell viability (Figure 3(a)), but significantly attenuated the H₂O₂-promoted apoptosis of H9c2 cells (Figure 3(b-c)). Besides, H₂O₂ treatment markedly declined the concentration of SOD (Figure 3(d)) and GSH-Px (Figure 3(e)) with the increased level of MDA (Figure 3(f)), which was notably reversed by medicated serum treatment not negative serum. In addition, medicated serum not negative serum signally dampened the H₂O₂-elevated the level of ROS (Figure 3(g)), while aggrandized the H₂O₂-lessened MMP level (Figure 3(h)) of H9c2 cells. Thus, these results indicated that the medicated serum of Xinshuaining preparation enhanced the cell viability and suppressed apoptosis and oxidative stress in H9c2 cells.

Figure 3. The medicated serum of Xinshuaining preparation elevated the cell viability and repressed apoptosis and oxidative stress in H9c2 cells. H9c2 cells were administrated with 75 μM H₂O₂ and 10% medicated serum. (A) The cell viability of H9c2 cells was detected by CCK-8. (B and C) The apoptosis rate was analyzed using flow cytometry assay. (D-F) The level of SOD, GSH-Px, and MDA was determined using commercial kits. (G) The ROS level was determined using a reactive oxygen species assay kit, and the fluorescence of the cells was measured by flow cytometry. (H) MMP was determined by JC-1 staining. The means ± SD of three independent samples are shown. *p < .05.

The medicated serum of Xinshuaining preparation regulated the PI3K/Akt/Nrf-2 signaling pathway

Next, the effect of the medicated serum on the underlying signaling pathway was further analyzed by western blot. The results displayed that the relative protein expression level of phosphorylated PI3K and Akt was memorably reduced with H₂O₂ treatment, which was rescued by the medicated serum treatment (Figure 4(a-b)). Also, the medicated serum treatment significantly enhanced the H₂O₂-induced diminishment of the protein expression level of Nrf-2 (Figure 4(a-b)). Therefore, these results suggested that the effect of the medicated serum of Xinshuaining preparation on H9c2 cells might be involved in the PI3K/Akt/Nrf-2 signaling pathway.

Figure 4. The medicated serum of Xinshuaining preparation modulated the PI3K/Akt/Nrf-2 signaling pathway. H9c2 cells were treated with 75 μM H₂O₂ and 10% medicated serum. (A) The protein level of PI3K, p-PI3K, Akt, p-Akt, and Nrf-2 was evaluated using western blot. (B) The relative intensity of the proteins is shown as a bar graph. The data were expressed after being normalized to β-actin or LaminA. The means ± SD of three independent samples are shown. *p < .05.

The medicated serum of Xinshuaining preparation enhanced the cell viability and reduced apoptosis and oxidative stress involved in the PI3K/Akt/Nrf-2 signaling pathway in H9c2 cells

To further confirm the effect of the medicated serum of Xinshuaining preparation on H9c2 cells involved in the PI3K/Akt/Nrf-2 signaling pathway, H9c2 cells were pre-treated with LY294002, a PI3K/Akt inhibitor, and then hatched with H₂O₂ and medicated serum. The medicated serum treatment observably increased the H₂O₂-reduced H9c2 cell viability, which was inverted by LY294002 pre-treatment (Figure 5(a)). On the contrary, LY294002 pre-treatment significantly promoted the decrease in the H9c2 cell apoptosis rate induced by medicated serum (Figure 5(b-c)). Besides, LY294002 pre-treatment prominently declined the medicated serum-enhanced level of SOD and GSH-Px, while elevated the diminishment of the MDA level induced by medicated serum (Figure 5(d-f)). In addition, LY294002 pre-treatment notably augmented the attenuation of the ROS level, whereas memorably dampened the aggrandizement of the MMP level induced by medicated serum (Figure 5(g-h)). Moreover, the medicated serum treatment markedly enhanced the H₂O₂-decreased relative protein expression level of phosphorylated PI3K and Akt, and Nrf-2, which was antagonized by LY294002 pre-treatment. No statistical differences were observed in the relative protein expression level of the PI3K and Akt among these five groups (Figure 5(i-j)). LY294002 pre-treatment alone significantly reduced the cell viability, the level of SOD and GSH-Px, the MMP level, and the relative protein expression level of phosphorylated PI3K and Akt, and Nrf-2, while prominently enhanced the apoptosis rate, MDA level, and ROS level (Figure 5(a-j)). Taken together, these findings indicate that the medicated serum of Xinshuaining preparation elevated the cell viability and diminished apoptosis and oxidative stress associated with the PI3K/Akt/Nrf-2 signaling pathway in H9c2 cells.

Figure 5. The medicated serum of Xinshuaining preparation increased the cell viability and suppressed apoptosis and oxidative stress related to the PI3K/Akt/Nrf-2 signaling pathway in H9c2 cells. H9c2 cells were pre-treated with 10 μM LY294002 for 10 min and then hatched with 75 μM H₂O₂ and 10% medicated serum. (A) The cell viability of H9c2 cells was determined by CCK-8. (B and C) The apoptosis rate was assessed by flow cytometry assay. (D-F) The level of SOD, GSH-Px, and MDA was detected using commercial kits. (G) The ROS level was analyzed using a reactive oxygen species assay kit, and the fluorescence of the cells was measured by flow cytometry. (H) MMP was evaluated by JC-1 staining. (I) The protein level of PI3K, p-PI3K, Akt, p-Akt, and Nrf-2 was examined by western blot. (J) The relative intensity of the proteins is displayed as a bar graph. The data were expressed after being normalized to β-actin or LaminA. The means ± SD of three independent samples are shown. *p < .05 vs. Control group; ^#p < .05 vs. H₂O₂ group; ^&p < .05 vs. H₂O₂+ medicated serum group.

Discussion

Oxidative stress is an important pathological process of a variety of cardiovascular diseases such as hypertension, atherosclerosis, myocardial hypertrophy, and heart failure (14). Xinshuaining preparation including a series of TCM has a therapeutic effect on heart failure (15). In the present study, the effect and the underlying mechanisms of Xinshuaining preparation on the H9c2 cells induced by H₂O₂ were explored. The results revealed that Xinshuaining preparation protected H9c2 cells from H₂O₂-induced oxidative damage via the PI3K/Akt/Nrf-2 signaling pathway.

A large number of studies have reported that TCM can prevent the heart from oxidative stress-induced damage. For instance, Shengmai injection relieves H2O2‑induced oxidative stress via suppressing ERK and activating AKT pathways in cardiomyocytes (16). [6]-Gingerol decreases the level of oxidative stress, inflammation, and apoptosis to alleviate myocardial fibrosis by inhibiting the TLR4/MAPKs/NF-κB signaling pathway (17). Wenxin particles ameliorate atrial remodeling via dampening oxidative stress and modulating mitochondrial homeostasis and function in diabetic rats (18). Similar to these previous findings, our results also exhibited that Xinshuaining preparation attenuated the oxidative stress in H9c2 cells, as indicated by the fact that the medicated serum of Xinshuaining preparation notably increased the H₂O₂-reduced concentration of SOD and GSH-Px, while dampened the H₂O₂-elevated level of MDA and ROS. SOD is an antioxidant enzyme that specifically scavenges oxygen-free radicals that exert a significant effect on the balance of oxidation and antioxidant and can disproportionate superoxide anion-free radicals to generate hydrogen peroxide to subsequently translate into water by GSH-Px (19). MDA is a degradation outcome of lipid peroxides, which represents the peroxidation degree of body fat (19). As the main free radical in the body, ROS can not only oxidize but also trigger a cascade reaction, thereby generating more ROS (20). Overabundance of ROS is the dominating precursor of oxidative stress (20). Therefore, Xinshuaining preparation inhibited the oxidative stress in H9c2 cells.

Evidence has revealed that excessive production of ROS perturbs cell function, which finally leads to irreversible cell damage and death (21). Our results consistently showed that H₂O₂ induced the reduction of cell viability, which confirmed the oxidative injury of H9c2 cells caused by H₂O₂. Increases of intracellular ROS promote the level of intracellular Ca²⁺, the expression of Bax-related genes, opening of mitochondrial permeability transition pore and activation of caspases, which eventually results in the cell apoptosis (20). Moreover, overproduction of ROS causes the reduction of MMP, which leads to the initiation of the mitochondrial apoptosis pathway (22). In the present study, the H₂O₂ treatment notably increased the apoptosis rate with the decreased MMP level of H9c2 cells. Furthermore, the medicated serum of Xinshuaining preparation significantly antagonized the changes induced by H₂O₂ treatment in H9c2 cells. Thus, these findings illustrated that Xinshuaining preparation protected H9c2 cells from oxidative stress-induced injury.

Nrf-2, a transcription factor, can bind to antioxidant response elements and modulate the expression level of a variety of antioxidant genes, and thus, it is tightly involved in the antioxidant response (23). The PI3K/Akt signaling pathway is prerequisite for the activation of Nrf-2 (24). The PI3K/Akt signaling pathway is a core signaling pathway that plays an important role in myocardial protection (25). Akt is a crucial downstream target protein of PI3K, which can be activated by phosphorylation of the PI3K/Akt signaling pathway to generate p-Akt. The previous study has shown that inducing cardiomyocytes to overexpress Akt or activate Akt can significantly resist hypoxia/reoxygenation damage of cardiomyocytes (26). Moreover, the PI3K/Akt signaling pathway can relieve cell apoptosis through reducing the expression of Bax gene, intracellular Ca²⁺ concentration, and caspase-3 activity via mitochondrial function (27). Also, the PI3K/Akt signaling pathway regulates the cell survival (28). Furthermore, the role of the PI3K/Akt/Nrf-2 signaling pathway in cardiac function has been reported in a series of studies (24,29,30). In addition, the role of the PI3K/Akt/Nrf-2 signaling pathway in oxidative stress has also been documented in diverse disease models (31–33). In the present study, the effect of the medicated serum of Xinshuaining preparation on the cell viability, apoptosis, and oxidative stress was notably antagonized by LY294002 pre-treatment. Therefore, we concluded that Xinshuaining preparation protected H9c2 cells from oxidative stress-induced injury via the PI3K/Akt/Nrf-2 signaling pathway.

In conclusion, we clarified that the medicated serum of Xinshuaining preparation increased the cell viability and suppressed apoptosis and oxidative stress. Mechanically, the medicated serum of Xinshuaining preparation enhanced the H₂O₂-induced diminishment of the protein expression level of phosphorylated PI3K and Akt, and Nrf-2. In brief, we hope that our results can offer a solid evidence base for clinical development and therapy of CVD.

Ethics approval and consent to participate

All the procedures were performed severely according to the National Institute of Health Guide for the Care and Use of Laboratory Animals. This study was also authorized by the Board and Ethics Committee of Affiliated Hospital of Chengdu University of Traditional Chinese Medicine.

Availability of data and materials

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions

MH, JL, and WX conceived and designed the experiments; HM, JL, and YY performed the experiments; MH, JL, YY, and YD analyzed the data; and WG, HF, and CW contributed reagents/materials/analysis tools. MH, JL, and WX wrote the manuscript. All the authors read and approved the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10641963.2022.2131806

Additional information

Funding

This work was supported by the Provincial Unit Traditional Chinese Medicine Development Fund (Center for the Prevention and Treatment of Major Diseases of Traditional Chinese Medicine) under Grant [number CYW2021070].