Ginsenoside Rg2 Attenuates Doxorubicin-induced Cardiomyocyte Apoptosis via PI3K/Akt Pathway

Doxorubicin (DOX) is an important drug for cancer therapy; however, its use is limited by its cardiotoxicity. Ginsenoside Rg2 is extracted from Panax ginseng, which is believed to have cardioprotective properties. However, to date, there have been no reports on whether ginsenoside Rg2 could protect cardiomyocytes against DOX. In this study, we investigated the action and underlying mechanisms of ginsenoside Rg2 upon DOX treatment. This study aimed to explore the cardioprotective effects of ginsenoside Rg2 against DOX treatment. Cell Counting kit-8 was used to determine cell viability and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining was used to detect apoptotic cells. Western blotting was used to investigate the relevant pathways. LY294002 (LY294), a PI3K inhibitor, was used in this study. We found that ginsenoside Rg2 signicantly (P < 0.01) neutralized cardiomyocyte apoptosis induced by DOX in a dose-dependent manner, which was blocked by LY294. Moreover, ginsenoside Rg2 upregulated Akt phosphorylation through the PI3K/Akt pathway and inhibited p53 expression. Taken together, Ginsenoside Rg2 attenuates DOX-induced cardiomyocyte apoptosis via the PI3K/Akt pathway. our showed p-Akt/t-Akt, was also suppressed by that the effect of Rg2 against DOX through the PI3K/Akt also


Introduction
Chinese herbal medicine (CHM), as a complementary and alternative medicine, has been used to treat heart conditions for thousands of years 1 . Ginseng, the root of Panax ginseng, is a kind of CHM widely used nowadays for its health bene ts and as a cardiovascular disease treatment in the United States 2 , Canada 3 , and Europe 4 . To date, over 100 kinds of ginsenosides have been identi ed 5 , and many of them have been studied for the treatment of various diseases [6][7][8][9][10] . Ginsenoside Rg2, a major component of the ginsenoside extracted from P. ginseng (Fig. 1). It has various pharmacological functions, such as antimyocardial brosis 11 , amelioration of vascular dementia 12,13 , improvement of glucose and fat metabolism 14 , and antioxidant 15 and antidepressant 16 effects. However, the effect of ginsenoside Rg2 on myocardial injury protection and its underlying mechanism remain unclear. Doxorubicin (DOX) is an effective drug for the treatment of malignant neoplasia. However, the application of DOX is limited by its cardiotoxicity. Previous studies have shown that long-term DOX application leads to high morbidity of irreversible and late-onset dilated cardiomyopathy 17,18 . Therefore, new strategies are needed to control DOX cardiotoxicity and protect cardiac myocytes from damage.
It has been shown that ginsenosides Rg1, Rg3, Rb1, and Rh2 19-22 could alleviate cardiotoxicity induced by DOX; however, there are no reports on ginsenoside Rg2. In view of previous studies, we hypothesized that ginsenoside Rg2 could have the same effect of attenuating DOX-induced cardiomyocytes apoptosis as other ginsenosides. To test this hypothesis, we examined whether ginsenoside Rg2 is functional in H9c2 cells treated with DOX. The expression of Akt and p53 was determined to demonstrate the possible mechanism of action. We aimed to determine whether Rg2 could protect the cells from apoptosis through the phosphatidylinositol 3-kinase (PI3K)/Akt/p53 signaling pathway.
We rst established a DOX-induced apoptosis model in cardiomyocytes. The cytotoxicity of DOX in H9c2 cells was analyzed using the CCK-8 assay, using the appropriate concentration of DOX (5 µM) at 24 h ( Fig. 2A). The cytotoxicity of ginsenoside Rg2 was also measured using the CCK-8 assay, which demonstrated that the maximum safe concentration was 300 µM after 24 h of treatment (Fig. 2B). Then we tested the effect of simultaneous ginsenoside Rg2 and DOX treatment in H9c2 cells showing that ginsenoside Rg2 signi cantly increased the cell viability in a dose-dependent manner (Fig. 2C). To further investigate the effect of simultaneous administration of ginsenoside Rg2 and DOX, H9c2 cells were pretreated with ginsenoside Rg2 (100, 200, and 250 µM) for 24 h before DOX (5 µM) treatment followed by TUNEL staining (Fig. 2D).
The results showed that ginsenoside Rg2 notably reduced the apoptotic rate compared with the DOX treated cells alone, and the apoptotic rate was even lower at 200 µM and 250 µM than at 100 µM ginsenoside Rg2 (P < 0.01) (Fig. 2E). These data suggested that ginsenoside Rg2 inhibited cardiomyocyte apoptosis against DOX.
As excessive ROS are generated by DOX, we then tested ROS content in H9c2 cells after treatment with ginsenoside Rg2 and DOX using the ROS assay kit. We found that, compared with the control group, a higher level of ROS was detected in the DOX (P < 0.01) and DOX + Rg2 + LY294 (P < 0.05) groups. Meanwhile, ROS was markedly reduced in the DOX + Rg2 group (P < 0.01) (Fig. 3A). These results suggest that ginsenoside Rg2 could reduce ROS generation caused by DOX.
To further clarify the mechanism by which ginsenoside Rg2 inhibits apoptosis and ROS generation in cardiomyocytes, LY294, an inhibitor of PI3K, was used in the treatment. ROS content was dramatically upregulated in H9c2 cells after DOX treatment, which was distinctly inhibited by ginsenoside Rg2. LY294 notably counteracted the inhibitory effect of ginsenoside Rg2 (Fig. 3A). We found that ginsenoside Rg2 signi cantly inhibited cardiomyocyte apoptosis induced by DOX, which was suppressed by LY294 ( Fig. 3B). These data suggest that the effect of ginsenoside Rg2 on cardiomyocyte apoptosis and ROS generation occurs through the PI3K/Akt pathway. Ginsenoside Rg2 upregulates Akt phosphorylation through the PI3K/Akt pathway and inhibits p53 expression.
To further reveal the mechanism of action of ginsenoside Rg2 against DOX, the PI3K/Akt/p53 pathway was studied. It was found that Akt did not signi cantly differ between the DOX and DOX + Rg2 groups, but p-Akt was both upregulated in the DOX group and DOX + Rg2 group, and the value of p-Akt/Akt was notably increased in H9c2 cells when treated with ginsenoside Rg2, which was inhibited by LY294 ( Fig. 4A, B). Meanwhile, DOX dramatically increased both p53 and p-p53 expression in H9c2 cells. Simultaneous ginsenoside Rg2 treatment signi cantly reduced p53 expression (P < 0.01) and although it did not signi cantly inhibit p-p53, a trend could be observed, although not within the function of LY294 (Fig. 4A, B). Taken together, these results indicate that ginsenoside Rg2 upregulates Akt phosphorylation through the PI3K/Akt pathway and inhibits p53 expression.

Discussion
Cancer is one of the main causes of death worldwide, and chemotherapy is an important treatment method. Although DOX is an important chemotherapeutic, it exhibits dose-dependent cardiotoxicity 18 . Although the detailed mechanism of DOX-induced cardiac toxicity has not been fully elucidated, numerous studies have indicated that DOX-related myocardial damage is possibly correlated with apoptosis 23 , in ammation, oxidative stress, and autophagy 18,24,25 . It has been reported that after the administration of DOX, excessive ROS are produced in cardiomyocytes, accompanied by the upregulation of p53 and caspase-3, thereby activating apoptotic pathways [26][27][28][29][30][31][32] , which eventually lead to cardiac dysfunction.
In our study, we found that DOX led to cardiomyocyte apoptosis and generated ROS at certain concentrations, which was consistent with previous studies. We also found that ginsenoside Rg2 signi cantly reduced cardiomyocyte apoptosis and ROS generation caused by DOX. Ginsenoside Rg2 is a ginsenoside found in ginseng. As a famous traditional Chinese medicine, ginseng is now widely used to reinforce human endurance and resistance to fatigue and physical stress, owing to its role in improving energy and longevity 33 . Many previous studies have demonstrated that ginseng plays a role in immunomodulation, cancer suppression, cardiac protection, and neuroprotection 34 . For instance, ginsenoside Rg3 can inhibit proliferation 35 , metastasis, and angiogenesis 36 , as well as activate apoptosis 37 and promote immunoreaction 38 . Ginsenosides, the main active ingredients of ginseng, have also been shown to have a cardioprotective function 5 . In terms of anti-apoptosis, ginsenoside Rb3 exerted anti-apoptotic effects on cardiomyocytes via the PPARα pathway 39 , whereas ginsenoside Rg1 attenuated dopamine-induced apoptosis in PC12 cells by suppressing oxidative stress 40 . Ginsenoside Rd was also reported to attenuate the mitochondria-dependent apoptotic pathway via Akt/GSK-3β signaling 41 . Ginsenoside Rg2 was previously reported to inhibit apoptosis induced by H 2 O 2 in H9c2 cells 42 and ameliorate chemical myocardial ischemia in rats 43 . In addition, in our study, the anti-apoptotic function of ginsenoside Rg2 in DOX-treated cells was further con rmed using TUNEL staining. This effect was dose-dependent. Ginsenoside Rg2 was also able to reduce ROS induced by DOX).
To further identify the mechanism of action of ginsenoside Rg2 against DOX, the PI3K/Akt pathway was investigated. The PI3K/Akt pathway is thought to be closely related to differentiation, proliferation, apoptosis, and oxidative stress. Its activation is known to reduce cardiomyocyte apoptosis 44,45 . p53, a downstream factor of the PI3K/Akt pathway, is a classic tumor suppressor and its activation has been reported to accelerate cardiomyocyte apoptosis [46][47][48] . Previous studies have also demonstrated that Akt phosphorylation and p53 inhibition could inhibit DOX-induced cardiomyocyte apoptosis and improve myocardial systolic function 49,50 . In this study, our data illustrated that the ginsenoside Rg2 inhibition of cardiomyocyte apoptosis induced by DOX was repressed by Ly294. Additionally, the results of our ROS assay were consistent with the CCK-8 assay, which showed that ginsenoside Rg2-induced suppression of DOX-induced ROS signi cantly inhibited Ly294. In addition, western blot analysis indicated that ginsenoside Rg2 notably upregulated p-Akt and p-Akt/t-Akt, which was also suppressed by Ly294. These results demonstrated that the effect of ginsenoside Rg2 against DOX was mediated through the PI3K/Akt pathway. Consistently, in a mouse model of myocardial infarction, ginsenoside Rg2 was also shown to improve cardiac function by promoting Akt phosphorylation 11 . Our results also showed that DOX signi cantly upregulated p53 expression, which was remarkably inhibited by ginsenoside Rg2. Although no signi cant difference was observed in the levels of p-p53 upon ginsenoside Rg2 treatment, there was a downregulation trend. However, it is unclear whether Ly294 represses the expression of p53 and p-p53 and elevates p-p53/p53. Our results demonstrated that ginsenoside Rg2 could downregulated DOXinduced p53 and p-p53, but the underlying mechanism that involves the PI3K/Akt pathway is still unclear, and further study is needed.
In conclusion, this study is the rst to report that ginsenoside Rg2 can potentially suppress DOX-triggered cardiotoxicity. Protection of ginsenoside Rg2 against DOX was shown to be associated with decreased production of ROS, downregulation of p53, and increased Akt phosphorylation. The attenuation of DOXinduced cardiomyocyte apoptosis triggered by ginsenoside Rg2 was mediated by the PI3K/Akt pathway. The underlying mechanism involving the PI3K/Akt pathway remains to be further explored. Given the antitumor effects of ginsenoside, it is hoped that the application of ginsenoside Rg2 in combination with DOX will improve cardiotoxicity and inhibit tumor cell activity at the same time. Cell culture.
H9c2 cells were cultured in DMEM containing 10% FBS and 1% P-S at 37 ℃ in a 5% CO 2 incubator. H9c2 cells were treated with 0.25% trypsin at 80% con uency and passaged at a 1:4 ratio approximately every three days. The cells used in subsequent experiments were in the logarithmic growth phase.
H9c2 cells were seeded in 96-well plates at a density of 1×10 4 cells/well and incubated at 37°C for 24 h.
To investigate the effect of ginsenoside Rg2 on DOX-induced cardiomyocyte apoptosis, H9c2 cells were rst treated with ginsenoside Rg2 for 24 h followed by the addition of DOX for another 24 h. Thereafter, the CCK-8 assay was performed. To determine the underlying mechanism, H9c2 cells were pre-treated with LY294 (1 µM), a PI3K inhibitor, for 30 min before ginsenoside Rg2 intervention. Apoptosis assay.
TUNEL staining was used to detect morphological features of apoptosis. After treatment with different concentrations (100, 200, and 250 µM) of ginsenoside Rg2 for 24 h, H9c2 cells were treated with DOX (5 µM) for another 24 h. Next, the cell supernatant was replaced with immune staining x solution for 30 min. After washing with PBS, the cells were mixed with enhanced immunostaining permeabilization solution for 5 min. The TUNEL solution was then added to the cells and incubated for 60 min at 37 ℃, followed by detection using a 200-fold uorescence microscope (Olympus BX50, Tokyo, Japan).

ROS assay.
The ROS assay kit was used to analyze ROS levels in cardiomyocytes. H9c2 cells were seeded in 96-well plates at a density of 1×10 4 cells/well. After intervention with ginsenoside Rg2, DOX, and LY294, DCFH-DA (10 µM) was added to the cells, followed by incubation for 20 min at 37 ℃. The cells were then washed three times with DMEM and ROS absorbance was measured at 488 nm using a multifunctional enzyme standard instrument.

Western blot analysis.
Brie y, the total proteins of H9c2 cells were extracted according to the manufacturer's instructions. The protein concentration was calculated using the BCA kit. After denaturation at 95°C for 5 min, proteins were separated via SDS-PAGE (10% separating gel and 5% stacking gel, 60 v for 30 min, 100 V for 1 h). The separated proteins were transferred to PVDF membranes at 350 mA for 1 h. Protein ladder markers were used to observe the molecular weight positions. The membranes were pre-incubated in blocking buffer-TBST containing 5% skim milk (w/v) for 1 h at room temperature (nearly 20°C) and then incubated overnight at 4°C with primary antibodies at a dilution of 1:1000. After washing with TBST, the membranes were incubated with a secondary antibody (1:2000 dilution) for 1 h at room temperature. Protein bands were detected using the ECL chemiluminescence system and a gel imaging system (Chemiscope 6300). ImageJ software was used for the gray value statistics. GAPDH was used as the loading control.

Statistical analysis.
All data are presented as mean ± standard deviation and were analyzed using SPSS Statistics 25. An independent sample t-test was used to compare the two groups. The comparison among the three groups (above) was performed via one-way analysis of variance and the least signi cant difference test was performed between groups. Non-normal distribution was represented by the median, and the rank sum test was adopted. Statistical signi cance was assumed if P reached a value < 0.05. Graphs were created using GraphPad Prism 7.0.

Data Availability
The data used to support the ndings of the study are included within the article.