Araloside C Prevent Myocardial Cell Apoptosis Through Regulating PI3K/AKt to Relieve Heart Failure

Background Araloside C (AsC), a natural saponin isolated from Aralia elata, has a wide range of anti-inammatory properties and has been found in recent years to have heart-protective effects. Present study aimed to determine the effects of AsC on myocardial cell apoptosis through regulating PI3K/AKt. Methods and Results Statistical analyses were performed using GraphPadPrism7.0 software. The differences between two groups and multiple groups were analyzed using t-test and one-way ANOVA, respectively. In vivo results showed that AsC administration could improve cardiac functions and apoptotic rate in HF model through PI3K/AKt signaling pathway, including increasing left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS), and decreasing left ventricular end systolic diameter (LVESD) and left ventricular end diastolic diameter (LVEDD) in detection of myocardial function, inhibiting LDH, CK, CK-MB, CK and HBDH in biochemical index level assessment, inhibiting BNP, ANG II, IL-1b, IL-4, IL-6 and TNF-a in immunological index level. ASC regulates the expression of key apoptotic molecules, including increasing the expression of Bcl-2 and Bax. ASC also regulates phosphorylation of p-PI3K and p-Akt. Conclusion This study suggested for the rst time that AsC could partially regulate the PI3K/AKt signaling pathway to prevent myocardial cell apoptosis. This study provided a basis for further research on effective substances in the treatment of HF.


Introduction
In recent years, the research on the treatment of heart failure (HF) with traditional Chinese medicine has attracted wide attention 1 . In particular, salvia miltiorrhiza, berberine, ginseng, astragalus and other traditional Chinese medicines, including salvianolic acid A, salvianolic acid B, tanshinone IIA, ginsenoside Re, berberine, avonoids, have been proved to improve cardiac hypertrophy, reduce cell apoptosis and in ammatory response, and protect heart function, has an important role [2][3][4][5] . Araloside C (AsC), a natural saponin isolated from Aralia elata, has a wide range of anti-in ammatory properties and has been found in recent years to have heart-protective effects. Luo et al studied on atherosclerotic mice, AsC can regulate macrophage polarization through sirt1-mediated autophagy, thereby reducing the formation of plaque area in atherosclerotic mice 6 . Moreover, inhibiting oxidative stress and Ca 2+ overload by regulating HSP90 can effectively reduce hypoxia/reoxygenation of H9c2 cardiomyocyte ischemia/reperfusion (I/R) injury 7-9 .
However, the therapeutic effect of AsC in rats with HF has not been reported so far, and its internal mechanism has not been reported. Therefore, we will focus on AsC to study its effect on cardiac function in rats with HF. Sun et al. found that the HSP90/Akt pathway induces cardiac hypertrophy and cell death, affecting the course of HF 10 . Therefore, we will study whether AsC can inhibit cell death, improve cardiac function and delay the occurrence and development of HF through the PI3K/AKt signaling pathway regulated apoptosis.

AsC repairs cardiac function in HF rats
In detection of myocardial function, we found that compared with Sham group, the heart function of rats in the HF group was signi cantly decreased, with LVEF ( Fig. 1C) and LVFS (Fig. 1D) . 4B), IL-1β (Fig. 4C), IL-4 ( Fig. 4D), IL-6 ( Fig. 4E), and TNF-α (Fig. 4F) compared to other groups (P < 0.05). There was no signi cant change between HF group and HF + PBS group. The expression of indicators decreased signi cantly after AsC intervention compared to HF and HF + PBS groups. Above all these results suggested that AsC treatment might decrease the expression of in ammatory factors to improve heart function in HF rats.

ASC inhibits cardiomyocyte apoptosis in HF rats
Apoptosis of HF rat cardiomyocytes was detected by TUNEL assay to evaluate the antiapoptotic effect of AsC on cardiomyocytes (Fig. 5). The results showed that compared with sham rats, the percentage of cardiomyocyte apoptosis in HF and HF + PBS rats was signi cantly increased. However, percentage of apoptotic myocardial cells was signi cantly lower after the intervention of the AsC, which indicated that the AsC had the effect of preventing the apoptosis of heart cells.
AsC promotes PI3K/Akt signaling pathway activation Total protein was extracted from cardiac tissue and WB was used to detect the changes in the levels of myocardial protein p/t-PI3K, p/t-Akt, Bcl-2, Bax, Caspase-3 and cytochrome C (Fig. 6A). WB analysis results (Fig. 6B) showed that compared with sham group, the expressions of P-PI3K, P-Akt and Bcl-2 were signi cantly reduced in the HF and HF + PBS groups and signi cantly increased after drug intervention. However, the expression of Bax increased compared with sham group, and decreased after AsC intervention. There were no statistically signi cant differences in the expression of other proteins between these groups.

Discussion
There are more than 4 million patients with HF in China, and the fatality rate is much higher than that in developed countries 13 . The research on the mechanism of HF has been in progress, and apoptosis has been proved to be helpful to the development of HF. Cardiomyocyte apoptosis occurs in a variety of cardiovascular diseases, such as myocardial infarction and I/R 14,15 . ASC is one of the most abundant triterpenoids isolated from A. elata, which has been clearly shown to stimulate cardiac activity 16,17 .
However, the cardioprotective effect of AsC and its mechanism remain unclear. In this study, we demonstrated that ASC could protect HF cardiomyocytes from apoptosis and inhibit the activation of Akt in HF myocardium. The results showed that AsC had a protective effect on myocardial cell apoptosis in rats with HF by inhibiting the overexpression of PI3K/Akt signaling pathway.
It has been reported that HF is not only due to the decrease of myocardial contractility, but also to the increase of apoptotic cells 19 . Apoptosis plays an important role in ventricular remodeling and HF 19,20 . Bcl-2 protein family determines the commitment of cells to apoptosis, and the activation of Caspase-3 triggers the execution of apoptosis. There are both pro apoptotic and anti-apoptotic proteins in Bcl-2 protein family. Bax promotes the formation of membrane pores in the form of oligomers, and releases pro-apoptotic substances into the cytoplasm to play a pro-apoptotic role, while Bcl-2 inhibits apoptosis by blocking the oligomerization of pro-apoptotic proteins 21 . The expression of Bcl-2, Bax, caspase-3 and cytochrome C were detected by Western blot. The balance between Bcl-2 and Bax was disrupted, while the expression of Caspase-3 and cytochrom C was not changed, suggesting that the apoptotic pathway was activated in the HF model. AsC treatment can increase the level of Bax, inhibit the expression of Bcl-2, but not affect the expression of Caspase-3.
In this study, the rat model of HF was established to explore the effects of PI3K/Akt signaling pathway on cardiac apoptosis. PI3K/Akt, as an important signal transduction pathway, plays an important role in cell survival, apoptosis and proliferation 21 .
The expression of p-PI3K and p-Akt increased in ASC treatment group, but decreased in HF rats. It suggests that AsC may play an anti-apoptotic role by regulating P-PI3K and P-Akt, rather than directly interacting with PI3K and Akt. Some ASC components have been con rmed to have anti apoptotic effect.
For example, quercetin, luteolin and tanshinone IIA have been shown to have anti-apoptotic effects on myocytes [22][23][24] . In future studies, we will verify the role of these potential active ingredients through in vitro and in vivo experiments. In conclusion, this study explored the protective effect of ASC in HF animal model and apoptosis model. The results showed that AsC could partially regulate the PI3K/Akt signaling pathway to inhibit myocardial cell apoptosis. This study provides a basis for further study of effective substances for the treatment of HF.

Establishment of the HF Model in Rats and Grouping
The HF model was established by abdominal aortic constriction. After feeding adult Wister male rats for 1 week, pentobarbital sodium anesthetized rats, and then the abdominal aorta was separated 1cm above the left renal artery through a median incision. Abdominal aorta was sutured with no. 22 needle 4-0 silk thread to form abdominal aortic stenosis (about 50% ~ 60%). Abdominal dissection was performed. At the same time, 1×105 U penicillin was intraperitoneally injected to prevent infection. The rats were monitored daily after surgery, and after 10 weeks, AsC medication interfered with HF (2.5 mg/kg/day) for 4 weeks.
The rats were divided into four groups (sham group, HF model, HF+PBS and HF+ AsC group) and ve of each group. Sham group rats received DMEM 70μl for control.
All rats were used for subsequent experiments in accordance with the Laboratory Animal Management Regulations and Animal Ethical Requirements before modeling. Animal experimental protocols were approved by the Ethics Committee of the Inner Mongolia People's Hospital and the study was carried out in compliance with the ARRIVE guidelines. Rats were sacri ced and anesthetized by intraperitoneal injection of pentobarbital sodium solution (1%) at a dose of 50mg/kg. We tried our best to reduce the number of animals that are used and reduce their suffering.

Detection of myocardial function
Ten weeks after the last administration, the rats in each group were anesthetized. After removing the chest hair and applying the coupling agent, the VisualsonicVevo 2100 imaging system was used to evaluate the preoperative and postoperative cardiac function through echocardiography. Mice were anesthetized with 2.5l /min iso urane before evaluation. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular fraction shortening (LVFS) and left ventricular ejection fraction (LVEF) were calculated with Vevo analysis software 25 .

Histological examination in rats
The rat heart was dissected, xed with 4% formalin for 24 hours, embedded in para n, and cut into 5μmthick slices. Then the slices were stained with Masson's trichrome staining (Solarbio, Beijing, China) and hematoxylin-eosin (HE) staining to observe heart tissue morphology. Image analysis software (Image-Pro Plus v4.0, Media Cybernetics, USA) was used to calculate the area occupied by collagen

Apoptosis Assay
According to the manufacturer's instructions, cardiomyocyte apoptosis was detected by TUNEL (EMD Millipore, Billerica, MA). The apoptosis of cardiomyocytes was brown. The average percentage of apoptotic cells in 5 randomly selected elds (under magni cation ×40) was calculated by Olympus microscope.

Western blotting (WB)
Total proteins were extracted from cardiac tissue and the levels of cardiac proteins P/t-PI3K, P/t-AKt, Bcl-2, Bax, Caspase-3 and cytochrome C were detected by WB.  Histological examination in rats.  AsC reduced plasma immunological index level.

Figure 6
Total protein was extracted from cardiac tissue and WB was used to detect the changes in the levels of myocardial protein.