The HuoXue DiTan Recipe Attenuates Left Ventricular Hypertrophy in Spontaneously Hypertensive Rats by Increasing Autophagy Through the PTEN/PI3K/AKT/mTOR Pathway

Hypertension-induced left ventricular hypertrophy (LVH) is associated with a reduction in autophagy, which can be inhibited by disruption of the PTEN/PI3K/AKT/mTOR pathway. The HuoXue DiTan recipe (HDR) is a commonly used prescription that has shown therapeutic effects on hypertension and its complications. However, its mechanisms are still unclear. In the present study, we hypothesized that HDR can regulate the PTEN/PI3K/AKT/mTOR signaling pathway and thereby reverse LVH by increasing autophagy in spontaneously hypertensive rats.


Introduction
Epidemiological studies have demonstrated that left ventricular hypertrophy (LVH) develops in >35% of hypertensive individuals and is a key risk factor for heart failure. Previous data have shown that reduction in LV mass lessens cardiovascular complications [1]. To more effectively prevent cardiac hypertrophy and more successfully apply therapeutic interventions, it is important to better understand factors involved in ventricular growth at an early stage of cardiac hypertrophy, rather than after it is established, so that therapeutic interventions can be more successfully applied [2]. However, the mechanism of hypertensioninduced LVH remains unclear.
In a recent study, we observed that profound changes in autophagy are present in the early stages of hypertension, before manifestation of LVH and cardiac dysfunction, in hearts of spontaneously hypertensive (SH) rats [3]. This sequence of events supports the concept of impaired autophagy preceding hypertension-induced LVH. Accordingly, targeting autophagy may offer a novel approach to preventing cardiac contractile dysfunction and structural remodeling in patients with hypertension [4]. Recent studies have shown that exercise-induced cardioprotection results from upregulation of autophagy in both acute and endurance exercise [5,6]. Over the last decade, much research has focused on identifying the signaling pathways that regulate cardiac hypertrophy [7]. Among these pathways, the mammalian target of rapamycin (mTOR) has emerged as a potentially important regulator of cardiac hypertrophy.
Besides increasing cell size, cardiac hypertrophy involves increased protein synthesis controlled by the PI3K/AKT/mTOR pathway [8]. AKT is a key regulator of cell growth and generally works via several downstream effectors such as mTOR, GSK-3β, and FOXO proteins, ultimately resulting in cardiac hypertrophy [9]. Phosphatase and tensin homolog on chromosome ten (PTEN) is a tumor suppressor protein and is considered to be a vital regulator of cell viability and apoptosis. PTEN also is a negative regulator of AKT signaling and reportedly suppresses cardiac hypertrophy [10]. PTEN inhibition in mice promotes cardiac hypertrophy and a marked decrease in cardiac contractility [11]. Interestingly, other studies [12] have revealed that traditional Chinese medicine (TCM) can enhance the expression of PTEN in myocardial, vascular, brain and kidney tissue, but the precise mechanism requires further investigation.
In China, TCM has attracted increasing attention for its associated multi-potent properties [13]. It is well known that Chinese herbal medicine is effective in amelioration of LVH. TCM can interact with various pathways to improve left ventricular hypertrophy (LVH) during hypertension-associated pathological changes [14]. Clinically, the HuoXue DiTan method, a TCM herbal compound, could lower blood pressure, improve oxidative stress and reverse LVH among hypertensive patients [15]. However, the speci c molecular mechanisms responsible for its effects have not been fully elucidated. Based on the evidence discussed above, the present study was designed to explore whether the HuoXue DiTan recipe (HDR) can alleviate LVH in spontaneously hypertensive rats by improving autophagy through the PTEN/PI3K/AKT/mTOR signaling pathway. Our research may provide a theoretical basis for the pharmacological effects of the HDR on vascular remodeling and left ventricular hypertrophy in patients with essential hypertension.

Materials
Animals and Treatments: The male WKY rats used in this study were purchased from the Chinese Academy of Sciences Shanghai Experimental Animal Center at 16 weeks of age with a body weight 300±20 g and a clean grade with certi cate: SCXK (Shanghai) 2003-0003. The male SH rats used in this study were purchased from Beijing Wei Tong Lihua Experimental Animal Center at 16 weeks of age with a body weight 300 ± 20 g and a clean grade with certi cate: SCXK (Beijing) 2007-2001. WKY rats and SH rats were housed until 21 weeks of age in a clean environment with a light/dark cycle of 12 h/12 h, relative humidity of 50-60%, ambient temperature 22-25℃, 4 rats per cage, and free access to food and water. All procedures were approved by the Animal Care and Use Committee of Central South University.
The SH rats were randomized into three groups of ten each: (1) The SH group was treated with distilled water and served as an untreated control, (2) the HDR group was treated only with HDR, 6.48 mg/kg/d, and (3) the inhibitor group was treated with both HDR, 6.48 mg/kg/d, and the PTEN inhibitor VO-OHpic, 10 mg/kg/d, via intraperitoneal injection every two weeks from the beginning of the experiment [16].
Every group was given daily oral lavage with the appropriate designated drug for 12 weeks. The WKY rats (WKY group) were also treated with distilled water as a blank control. Rat body weight, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured each day for two weeks, and systolic cardiac function was monitored via echocardiography twice, at the beginning and end of the experiment. All rats survived the study, and no signi cant abnormalities were observed.
Diethylpyrocarbonate (DEPC) and PCR primers were from Beijing Parkson Gene Technology Ltd, China. dNTP, RNAsin, Taq DNA polymerase, and DNA ladder were from Sino-American Inc, Beijing, China. The primary antibodies were from ThermoFisher Scienti c Inc., USA. The malondialdehyde (MDA) assay kit (A003-1-2), nicotinamide adenine dinucleotide phosphate (NADPH) kit (A127-1-1), and superoxide dismutase (SOD) kit (A001-3-2) were purchased from the Nanjing Jiancheng Biology Engineering Institute. The TUNEL apoptosis detection kit (C1086) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The CFX 96 touch uorescence quantitative PCR detection system was obtained from Bio-Rad (United States), and the LSM510 laser confocal microscope from Carl Zeiss (Germany). HDR consists of Salvia miltiorrhiza, 15 g, sappanwood, 10 g, Pinellia ternata, 10 g, tangerine peel, 10 g, Poria cocos, 9 g, and licorice root, 5 g. The Pharmacy Department of the Xiangya Hospital of Central South University purchased, mixed, decocted, ltered, concentrated and dried the six herbs and extracted the powder according to standard procedures. Finally, we obtained 3.75 kg powder containing 6.48 g of crude medicine per gram of powder. Brie y, according to the Chinese Pharmacopoeia (2015 edition), tanshinone A and protosappanin A were quanti ed for quality control. Their yield was 0.35 and 0.42 mg/g, respectively, as measured by high performance liquid chromatography (HPLC). Standard samples of tanshinone A and protosappanin A were purchased from Chengdu Gelipu Bio-tech Co., Ltd.(Chengdu, China).

Measurement of tail artery blood pressure
Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured weekly in the tails of conscious rats using a noninvasive computerized tail-cuff system (Kent Scienti c Corporation, CT, USA).
The rats were warmed at 28°C for 10-15 min before the measurements to allow for detection of tail artery pulsations and to achieve a steady pulse. To minimize stress-induced uctuations, the rats were pre-trained by measuring blood pressure daily for at least 1 week before the experiments began. Tail artery blood pressure was averaged over ve measurements.

Echocardiography
After the 12-week drug administration period, transthoracic echocardiography was performed under iso urane anesthesia using an ultrasound system (Vevo 2100, VisualSonics, Toronto, Canada) with a 21 MHz probe. LV mass and LV volume during diastole (LVVd) and systole (LVVs) were measured. The LV mass-to-body weight ratio and ejection fraction (EF) were calculated [17]. Measurements were averaged over three consecutive cardiac cycles.

Blood sample analysis
At the end of the observation, blood samples were collected. Serum samples were obtained by centrifugation of the blood samples at 3000 rpm for 10 min at 4°C. The supernatant was collected and immediately stored at −80°C, and thawed at −4°C before analysis. The serum levels of MDA and SOD, and the activity of NADPH oxidase in myocardial tissue, were determined using the appropriate biochemical kits.

Measurement of left ventricular mass index
When all measurements were complete, the chest of the rat was immediately opened and the heart quickly removed. After drying with lter paper, the remaining septal and left ventricular free walls were weighed to determine left ventricular mass. The left ventricular mass index (LVMI) was calculated as left ventricular mass/body mass (mg/g). The central section of the left ventricle was harvested and xed in 10% neutral formalin, and the remaining portion was snap frozen in liquid nitrogen and stored at −80℃.

Histological analyses
(1)Hematoxylin-eosin staining Heart sections (5 µm thick) were examined by hematoxylin-eosin (HE) staining (Service Biological Technology Co., Ltd, Wuhan, China) to measure the cross-sectional area of cardiomyocytes. Three to ve random elds (around 30-50 cells per eld) were selected from each of three sections from each animal for observation under a light microscope (Olympus Corporation, Tokyo, Japan).
(2)Masson's trichrome staining The sections were placed in composite Masson's staining solution for 5 min, washed in a 0.2% acetic acid solution for 1 min, stained with 5% phosphotungstic acid for 5 min, dipped in 0.2% acetic acid solution for 2 min, stained with brilliant green staining solution for 5 min, dipped in 0.2% acetic acid solution twice, separated in 95% alcohol, dehydrated in gradient alcohol, cleared with xylene, and mounted with neutral gum. Masson's trichrome stained myocardial cells red and collagen green. Collagen content was observed under a light microscope, and 10 random views were analyzed using image analysis software (Image-Pro Plus, Media Cybernetics, USA).

(3)Transmission electron microscopy
Left ventricular tissue was cut into cubes of approximately 1 mm on a side and xed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer overnight at pH 7.4 and 4°C. Subsequently, the sections were put into 1% osmium tetroxide for 2 hours and then dehydrated in a graded ethanol series. After embedding in epoxy resin, ultra-thin sections (60-70 nm) were post-stained with uranyl acetate and lead citrate. Finally, the sections were analyzed using a JEM-1010 transmission electron microscope (JEOL Ltd, Tokyo, Japan).
(4)TUNEL assay Three para n sections randomly picked from left ventricular tissue were analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay according to the manufacturer's instructions as previously described [18]. The TUNEL index (%) was calculated as the ratio of the number of TUNEL-positive cells divided by the total number of cells. At least 3 representative elds were evaluated from each experimental group, from which an average value was calculated.

(5)Immunohistochemical staining
The myocardial tissue samples were embedded in para n and sliced into 4 µm-thick sections. After being depara nized and rehydrated, sections were blocked with 5% goat serum for 1.5 h, and incubated overnight at 4°C with β-myosin heavy chain (β-MHC) monoclonal antibodies (1:300, Abcam Technology, Inc. Cambridge, MA, USA). After washing with PBS, a secondary antibody were sequentially incubated at 37°C for 45 min, washed with PBS, and stained with diaminobenzidine (DAB) (K5007, DAKO, Germany). Cells in 6 random elds of each section were counted at ×400 magni cation. The optical density (OD) of positive cells was analyzed by Image-Pro Plus 6.0 software. The average integrated optical density (AIOD) was calculated as follows: AIOD = positive area × OD/total area.

Quantitative real-time PCR
Total tissue RNA was puri ed from hearts using Trizol (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the GoScript™ reverse transcription system (Promega, Southampton, UK). Quantitative real-time PCR (qPCR) was performed using SYBR Green Master Mix (Takara, Kusatsu, Japan) on an Applied Biosystems 7500 Fast System (ABI, Carlsbad, CA, USA) as described previously [19]. The genespeci c primer sequences (TaKaRa Bio) are shown in Table S1. GAPDH was used as an internal control. The primers were obtained from Sangon Biotech (Shanghai, China). Relative mRNA levels were calculated using the 2 ∆∆Ct method as described previously [19].

Western blotting
Total protein was extracted from heart tissues using lysis buffer containing protease/phosphatase inhibitors (Thermo Fisher Scienti c, Carlsbad, CA, USA). Protein concentrations were assessed using the Abbkine Protein Quanti cation Kit (Thermo Fisher Scienti c, Carlsbad, CA, USA). Equal amounts of proteins (40-50 µg) were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene uoride) membrane (Millipore, Billerica, MA, USA). Immunoblotting analysis was performed as described previously [20]. All protein levels were normalized to GAPDH. Images were captured and quanti ed by FluorChem M (ProteinSimple, San Jose, CA, USA). The primary antibodies are listed in Table S2. After washing with TBST (tris buffered saline with tween), secondary antibody (1:200) was added, and the blots were incubated at room temperature for 3 h before again being washed with TBST. ECL (enhanced chemiluminescence) developing solution was added to the membranes, which were then exposed to X-ray lm for 5-8 min. Quantity One analysis software (Bio-Rad, Hercules, CA) was used to analyze the results, and the integrated optical density (IOD) value was obtained by dividing the level of the target protein by that of the internal reference band (GAPDH) and used as the nal result for statistical analysis.

Statistical analyses
Data are presented as mean ± standard error of the mean (SEM). Using GraphPad Prism 4.0 (GraphPad software Inc., CA, USA), statistical signi cance among multiple groups was evaluated by one-way analysis of variance (ANOVA) with the Bonferroni post hoc test. A two-tailed P value < 0.05 was considered statistically signi cant.

Results
(1) Administration of HDR attenuated hypertension and cardiac dysfunction in SH rats As shown in Fig. 1A, SBP and DBP increased signi cantly in the SH group compared with the WKY group throughout the experiment (P < 0.01); Compared with the SH group, the HDR group had lower SBP and DBP over a period of 4-12 weeks (P < 0.05). However, when the PTEN inhibitor was added to the TCM intervention (HDR-treated) group, systolic and diastolic blood pressure increased signi cantly over the same period (P < 0.05). Echocardiographic parameters for these treatments are shown in Figure 1B-D.
However, when the PTEN inhibitor was added to treatment of the TCM intervention group, there was a signi cant decrease in these differences between the HDR-treated and inhibitor groups (LVPWd, P < 0.05; IVSd, P < 0.05; LVDd, P < 0.01; LVD.vol, P < 0.05; and LVEF, P < 0.05). To further con rm the role of HDR in SH rats with ventricular hypertrophy, mRNA expression of the hypertrophic markers β-MHC and ANP was detected by qRT-PCR, and β-MHC protein expression was assessed by immunohistochemical analysis (Fig. 2). Expression of ANP and β-MHC signi cantly increased in the SH group compared with the WKY group (P<0.01), while they signi cantly decreased in the HDR group compared with the SH group (P<0.01), and this was largely reversed by the PTEN inhibitor for both ANP (P<0.05) and β-MHC (P<0.01). These results suggest that HDR markedly improved blood pressure and cardiac function in SH rats, and that the observed effects may be associated with the PTEN signaling pathway (2) Administration of HDR alleviated left ventricular hypertrophy and brosis in SH rats Coronary sections of the heart are shown in Fig. 3A-B. Compared with WKY rats, SH rats had signi cantly higher heart size (P<0.05), heart weight (HW)/body weight (BW) ratio (P<0.01), and HW/tibial length (TL) ratio (P<0.01). The ratio of HW/BW and HW/TL signi cantly decreased in the HDR group compared with the SH group (P<0.01, P<0.05), which was largely reversed by treatment with the PTEN inhibitor (P<0.05). Histopathological analysis using HE (Fig. 3C) and Masson's trichrome (Fig. 3D) staining revealed that cardiomyocyte area and collagen deposition were higher in SH rats than in WKY rats (P<0.01) (Fig. 3E-F). However, the two quantities signi cantly decreased in the HDR group compared with the SH group (P<0.01, P<0.05), which was largely reversed by the PTEN inhibitor (P<0.01). These results show that HDR markedly reduced ventricular hypertrophy and brosis in SH rats, and that the observed effects may be associated with the PTEN signaling pathway.
(3) HDR attenuates oxidative stress in SH rats with ventricular hypertrophy.
ELISA was used to detect the level of SOD and MDA in serum, and the activity of NADPH oxidase in heart tissue. The activity of NADPH oxidase was signi cantly greater in the SH group than in the WKY group (P<0.01; Fig. 4D). The levels of SOD and MDA were signi cantly less in the SH group than in the WKY group (P<0.01). Following HDR treatment, the activity of NADPH oxidase signi cantly decreased, and SOD and MDA levels signi cantly increased, over those observed in the SH group (P<0.05, P<0.01). Following treatment with PTEN inhibitor, the effect of HDR was reduced. These results suggested that following treatment with HDR, cardiomyocyte antioxidative activity increased and oxidative stress injury in ventricular hypertrophy was attenuated.
(4) HDR increased autophagy and prevented apoptosis in SH rats with ventricular hypertrophy.
An increase in apoptosis in pathological cardiac hypertrophy was observed in SH rats in the present study. Results of the TUNEL assay ( Fig. 4A-B) showed that in comparison with WKY rats, the percentage of TUNEL-positive cells was higher in SH rats, which was accompanied by an increase in expression of the apoptosis-related proteins Bcl-2, p53 and caspase-12. Treatment with HDR for 12 weeks reversed the increase in the Bcl-2, p53 and caspase-12 expression in SH rats and reduced the percentage of TUNELpositive cells. Following treatment with the PTEN inhibitor, the effect of HDR was reduced. These results indicate that HDR attenuates cardiomyocyte apoptosis in SH rats with ventricular hypertrophy. Next, we wanted to con rm that autophagy was in fact upregulated after HDR treatment in ventricular hypertrophy ( Figure 4E). Expression of autophagy-related proteins was assayed ( Figure 4F-G). SH rats exhibited a signi cant downregulation of Beclin 1 and LC3 /LC3 expression (P <0 .05) and upregulation of p62 expression (P < 0.01) compared with WKY group. However, the HDR-treated groups showed upregulation of Beclin 1 and LC3 /LC3 expression (P <0 .05) and downregulation of p62 expression (P < 0.01) compared with the SH group. Following treatment with the PTEN inhibitor, the effect of HDR was reduced. These results suggest that HDR markedly increased autophagy and prevented apoptosis in SH rats with ventricular hypertrophy, and that the observed effects may be associated with the PTEN signaling pathway.
To identify the mechanism by which HDR increased autophagy in SH rats with ventricular hypertrophy, expression of PTEN mRNA and protein was detected (Fig. 5A-C). PTEN expression signi cantly decreased in the SH group compared with the WKY group (P<0.01). In addition, the HDR-treated group exhibited upregulation of PTEN expression (P <0 .05) compared with the SH group. Following treatment with the PTEN inhibitor, the effect of HDR was reduced. Furthermore, we measured expression of PI3K/AKT/mTOR signaling pathway-associated proteins, including PI3K, AKT and mTOR, in rat myocardial tissue (Fig. 5E-F). Expression of the phosphorylated proteins, p-PI3K, p-AKT and p-mTOR, signi cantly increased in the SH group compared with the WKY group (P<0.05, P<0.01, respectively). In addition, the HDR-treated group exhibited downregulation of p-PI3K, p-Akt and pmTOR expression (P <0 .05) compared with the SH group. Following treatment with the PTEN inhibitor, the effect of HDR was reduced. These data suggest that the PTEN/PI3K/AKT/mTOR signaling pathway plays a key role in autophagy in SH rats with ventricular hypertrophy.

Discussion
The exact mechanism by which hypertension results in ventricular hypertrophy remains unclear. The molecular mechanisms of ventricular hypertrophy involve multiple factors, including accumulation of reactive oxygen species, induced apoptosis, DNA damage, and mitochondrial dysfunction [21]. Autophagy dysfunction plays an important role in ventricular hypertrophy resulting from essential hypertension [22]. The aim of the present study was to elucidate the impacts of the HDR on ventricular hypertrophy in SH rats by examining autophagy. Furthermore, we examined the potential cardioprotective effect of the HDR via inhibition of the PTEN/PI3K/AKT/mTOR signaling pathway, increased autophagy and prevention of oxidative stress in SH rats. Our data demonstrated that oral administration of the HDR could attenuate pressure overload-induced ventricular hypertrophy via inhibition of the PTEN/PI3K/AKT/mTOR signaling pathway.
Cardiac remodeling is one of the most common complications associated with hypertension, and it is likely to be a risk factor that is independent of blood pressure [23]. At the age of 16 weeks, in comparison with age-matched WKY rats, SH rats exhibited signi cant myocardial hypertrophy and remodeling with abnormal heart function. Previous studies have shown that oxidative stress plays an important role in left ventricular hypertrophy with hypertension [24]. MDA and SOD are markers of oxidative stress, and NADPH oxidase is related to reactive oxidase stress in activated RAAS [25,26]. In the present study, the ELISA assay revealed that the activity of NADPH oxidase signi cantly increased, the levels of SOD and MDA signi cantly decreased, and expression of ANP and β-MHC, indicators of pathological cardiac hypertrophy, increased in cardiomyocytes in SH rats as compared with WYK rats. HDR administration to SH rats reduced blood pressure, improved EF, reduced heart weight and cardiomyocyte cross-sectional area, inhibited oxidative stress, and downregulated gene expression of myocardial hypertrophy markers (ANP and β-MHC). These results strongly suggest that HDR treatment improved heart function and reduced cardiac hypertrophy associated with hypertension.
HDR is derived from a classic prescription of TCM. Previous studies have shown that TCM (Salvia miltiorrhiza, hematoxylin, Pinellia ternata and Poria cocos) can signi cantly improve left ventricular hypertrophy caused by hypertension through a variety of genes and signaling pathways [27][28][29][30]. To explore the mechanism by which HDR improves ventricular hypertrophy caused by hypertension, we studied genes and signaling pathways related to cardiomyocyte apoptosis and autophagy in ventricular hypertrophy. In addition, the TUNEL assay revealed that the hearts of SH rats contained greater numbers of apoptotic cells. To determine the molecular mechanism of these changes, we examined gene markers for apoptosis, anti-apoptosis, and cell survival. In SH rats, expression of the genes for the proapoptotic markers p53 and Bcl 2 was upregulated. Interestingly, we found that treatment of SH rats with HDR resulted in a signi cant reduction in expression of these proapoptotic markers, leading to increased cardiomyocyte survival, consistent with previous observations [31]. Caspase 12 expression plays an important role in mitochondria-dependent apoptosis [32]. In this study, SH rats exhibited upregulated caspase 12 protein expression. In contrast, the HDR-treated SH group exhibited reduced caspase 12 protein expression. These data demonstrated that HDR could attenuate cardiac apoptosis by inhibiting expression of survival proteins.
Autophagy is a highly conserved and ubiquitous metabolic pathway in living organisms [33]. Previous studies have demonstrated that Beclin 1 is the earliest self-localizing gene in the structure of autophagic precursors and is believed to regulate other autophagic genes, cardiac-speci c overexpression of Beclin-1 promoted autophagy, and improved cardiac function [34,35]. We observed a dramatic decrease in LC3II and Beclin 1 in LVH of SH rats. In addition, 12 weeks of HDR treatment induced activation of the autophagic pathway in SH rat ventricles, including enhanced expression of Beclin 1 and an increased LC3-II/LC3-I ratio. Knock-out or silencing of the p62 gene in cardiomyocytes decreased autophagic activity [36]. We then measured expression of p62 and found that its gene was also obviously upregulated in LVH. In contrast, we found that the expression of p62 was signi cantly downregulated at the protein level in the left ventricles of rats with HDR-induced myocardial hypertrophy. These ndings are consistent with previous results demonstrating that autophagy is enhanced by TCM [37], and that Salvia miltiorrhiz provides cardioprotection by upregulating autophagy [38]. Taken together, these results suggest that HDR can increase autophagy and attenuate apoptosis in cardiomyocytes, which play an important role in pressure-overload cardiac hypertrophy.
Regulation of autophagy is rather complicated. The key players in initiation of autophagy are mTORrelated targets [39] regulated by signaling pathways such as the PI3K/AKT pathway [40], which is the main regulator of cell growth and survival [41] and plays an important role in survival of cardiomyocytes. Activation of the PI3K/Akt pathway phosphorylates mTOR, thereby inhibiting autophagy [42]. The PI3K/Akt pathway is one of the most important upstream pathways of mTOR [43]. Poria cocos, Glycyrrhiza uralensis and Salvia miltiorrhiza, the main ingredients of HDR, can upregulate autophagy through the PI3K/Akt pathway signal [44][45][46].
Given the above relationships, we focused our attention on mTOR, the major regulator of autophagy in cells. TCM treatment of different cell types has shown a clear decrease in the level of p-mTOR [47,48].
Thus, mTOR may be a direct target of TCM, similar to the effect of rapamycin. However, upstream of mTOR, a sharp decrease in the expression of both active forms of AKT, p-AKT (S473) and p-AKT (T308), is observed in response to TCM [49]. These observations led us to believe that downregulation of this pathway may be targeted through PTEN, a natural inhibitor of the PI3K-AKT pathway [50]. PTEN is a tumor suppressor gene that can induce autophagy through the PI3K-AKT pathway [10]; in contrast, its suppression can inhibit autophagy [51]. Our data also suggested a major role for PTEN in HDR-induced autophagy in SH rats, wherein HDR treatment led to greatly enhanced expression of PTEN in myocardial tissue, resulting in suppression of the AKT pathway and induction of autophagy. This nding is consistent with our observation that HDR exhibits a similar mechanism related to induction of autophagy in different cell types [52]. To con rm the involvement of PTEN in HDR-induced autophagy, we attempted to knock down expression of PTEN with an inhibitor (VO-OHpic). Interestingly, HDR could increase cell autophagy in SH rats, as indicated by a sharp increase in autophagic ux. However, HDR did not elicit the autophagic response in the PTEN inhibitor group, consistent with the observations above [53]. Thus, we conclude that PTEN-mediated inhibition of the PI3K/AKT/mTOR pathway plays a role in induction of autophagy by HDR. We only investigated the effects of HDR on signaling pathways, but not its in uence on blood vessels, which are important sites of changes leading to cardiac remodeling. We expect that additional mechanisms of HDR treatment will be investigated in future experiments.

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

Competing interests
The authors have declared that no competing interest exists.   The effect of HDR on the hypertrophic marker gene and protein expression in rats with ventricular hypertrophy. A: Immunohistochemical analysis of β-MHC in myocardial tissue (brown indicates positive cells, ×200). B: Semiquantitative rank scores for features of β-MHC-positive cells in myocardial tissue. C: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression of ANF and β-MHC. *P<0.05, **P<0.01 compared with the WKY group; # P<0.05, ##P<0.01 compared with the SH group; §P<0.05, § §P<0.01 compared with the HDR group.