Introduction
Obstructive sleep apnea (OSA) is a sleep breathing disorder characterized by intermittent upper airway collapse during sleep and leads to sleep fragmentation [
1]. Obstructive sleep apnea is often associated with cardiovascular diseases including coronary artery diseases and congestive heart failure [
2,
3]. Noninvasive mechanical ventilation therapy reduced left ventricular end systolic and diastolic diameters, reduced the occurrence of cardiovascular complications in coronary heart disease with OSA [
4]. Chronic intermittent hypoxia (CIH) showed various long-term cardiovascular pathophysiologic outcomes during sleep which are similar to OSA [
1]. Chronic intermittent hypoxia was often used in animal models to simulate the experiment of obstructive sleep apnea [
5,
6].
Rhodiola Crenulata, a species of Rhodiola, was often applied to avoid altitude sickness [
7,
8] and it have been reported to decreased hypoxia-induced oxidative stress [
8,
9]. In general, Rhodiola increased stress resistance, cardiopulmonary protection, and cardiovascular function [
7]. Rhodiola rosea, a species of Rhodiola, as an adaptogen, have effects of anti-fatigue, anti-stress, anti-hypoxic, anti-cancer, and antioxidant in cell cultures, animals, or humans [
10,
11]. Salidroside [2-(4-hydroxyphenyl)ethyl beta-D-glucopyranoside], a chemical component of Rhodiola Crenulata with phenylpropanoid glycoside was regarded as an active ingredient of Rhodiola [
12]. In general, salidroside have been reported to protect cardiomyocytes against cardiotoxicity [
13], stimulate mitochondrial biogenesis, protect endothelial dysfunction [
14], enhance stem cells [
15], induce erythropoietin [
16]. In our previous study, salidroside showed protection through chronic intermittent hypoxia-induced Fas-dependent and mitochondria-dependent apoptotic pathways on mice hearts [
5]. However, the effect of Rhodiola Crenulata on cardiovascular health is still unclear.
The apoptosis decreases cardiomyocytes in cardiomyopathies and shows also to be a predictor of cardiac diseases or heart failure [
17]. From our previous studies, cardiac apoptosis were found in obesity [
18,
19] smoking exposure [
20], and under long-term but not short-term intermittent hypoxia [
6,
21,
22]. The Fas receptor-dependent and mitochondria-dependent apoptotic pathways are the two major pathways triggering cardiac apoptosis [
23]. The Fas receptor-dependent apoptotic (Type I) pathway is a key pathway triggering cardiac apoptosis [
23]. The Fas binding the Fas receptor [
23‐
25], trailed by Fas-receptor oligomerisation results in the death-inducing signal complex, begins with recruitment of the Fas-Associated Death Domain (FADD) adaptor protein [
23]. The FADD activates caspase 8, cleaves pro-caspase 3 and undergoes autocatalysis forming active caspase 3, an effector caspase of apoptosis [
24,
25].
The mitochondria-dependent (Type II) apoptotic pathway begins with the apoptosis-regulating protein Bcl-2 family, including anti-apoptotic Bcl-2 and Bcl-xL as well as pro-apoptotic Bad and Bax [
23,
25,
26]. Pro-apoptotic Bcl2 family augments cytochrome
c release from mitochondria [
23,
25,
27]. Cytochrome
c release into cytosol activates caspase-9, then caspase-3 completes the apoptotic program [
23,
25]. Additionally, t-Bid was considered to be a key intracellular molecule signaling mediator from Fas to mitochondrial pathway since activated caspase 8 can cleave Bid to t-Bid then release cytochrome
c to mitochondria-dependent apoptosis activity [
23,
24]. In our previous studies, 8-week chronic intermittent hypoxia activated the Fas-dependent and mitochondria-dependent apoptotic pathways and increased cardiac apoptosis in rodent hearts [
5,
6,
21,
22].
Vascular Endothelial Growth Factor (VEGF), a potent angiogenesis angiogenic growth factor, promotes survival, reduces Endothelial Cells apoptosis and activates PI3K/Akt signaling in human endothelial cells [
28]. VEGF up-regulates pro-survival Akt/Bcl-2 signaling pathways as a survival factor for protecting cardiomyocyte against apoptosis [
29]. Akt, as a key part in the phosphatidylinositide 3-kinase (PI3K) pathway, a vital spot regulatory kinase through pro-survival and protective effect in myocardium [
30].
The effect of Rhodiola Crenulata on cardiac apoptosis in severe sleep apnea i.e. under chronic intermittent hypoxia is not understood. In the study, we investigate whether Rhodiola Crenulata have protective effects on mice heart with chronic intermittent hypoxia. We hypothesized that Rhodiola Crenulata may prevent cardiac apoptosis via Fas-dependent and mitochondrial-dependent anti-apoptotic pathways and VEGF-related pro-survival pathways in chronic intermittent hypoxia.
Materials and Methods
Animal model
The study was performed on sixty-four 5–6 months old C57BL/6 J mice were divided into four groups i.e. Control group (21 % O2, 24 h per day, 8 weeks, n = 16); Hypoxia group (Intermittent hypoxia: 7 % O2 60 s and alternating 20 % O2 60 s, 8 h per day, 8 weeks, n = 16); Hypoxia + 90RC and Hypoxia + 270RC group (Intermittent hypoxia for 1st 4 weeks; Intermittent hypoxia pretreated 90 mg/Kg and 270 mg/Kg Rhodiola Crenulata by oral gavage per day for 2nd 4 weeks, each n = 16). After exposure with normoxia or hypoxia and saline or RC, the hearts were excised and analyzed by heart weight index, H&E staining, TUNEL-positive assays and Western Blotting. Ambient temperature was kept at 25 °C and the animals were exposed to an artificial 12-h light–dark cycle, the light period which began at 7:00 am. Mice were provided with standard laboratory meals (Lab Diet 5001; PMI Nutrition International Inc., Brentwood, MO, USA) and water ad libitum. Protocols were proved by the Institutional Animal Care and Use Committee of China Medical University, Taichung, Taiwan.
Rhodiola crenulata
Rhodiola Crenulata was provided by TCM Biotech International Corp (Taiwan). The extraction process was as follows: The raw material was analyzed and verified to be free of organic toxins and heavy metals (Pb, Hg, Cd, As, Cu) or below acceptable levels. The raw material was ground and sifted with a 20 mesh screen. The grainy powder was extracted and compressed then freeze dried to reduce water content to less than 8 %. The material was then ground to return to a powder form (water less 6 %). The powder was sifted then packaged in a sterile aluminum bag.
Echocardiography
The trans-thoracic echocardiographic images of all mice were obtained using Philips M2424A ultrasound systems (Andover, MA) in anesthesia with 1 % isoflurane through a nose cone. The M-mode echocardiographic inspection was executed applying a 6–15 MHz linear transducer (15–6 L) through a parasternal long axis approach. The left ventricular M-mode dimensions on the stage of the papillary muscles contained inter-ventricular septum (IVS), left ventricular internal end-diastolic dimensions (LVIDd), left ventricular internal end-systolic dimensions (LVIDs), posterior wall thicknesses (LVPW), and fractional shortening (FS). FS% was calculated as a result of the equation FS% = [(LVIDd-LVIDs)/LVIDd]/100.
Cardiac characteristics
The hearts of mice in the four groups were extracted and cleaned with PBS. The weights of the left ventricles and the whole heart were measured. Once weighting, the 6 hearts of mice in each group were soaked in formalin and added analyzed by Hematoxylin-eosin, Masson trichrome staining, DAPI staining and TUNEL assay. The other 6 hearts of mice in each group were cleaned, frozen, and added analyzed by Western Blotting. Besides, right tibia lengths were measured using a digital caliper and the left heart weight were weighed. The whole heart weight (WHW) to body weight (BW) ratio, the left ventricle weight (LVW) to body weight (BW) ratio, the left ventricle weight (LVW) to the to the whole heart weight (WHW) ratio, and the whole heart weight to tibia length and the left ventricle weight to tibia length ratio were determined for analysis.
The left ventricle samples of cardiac tissue extracts were homogenized in a lysis buffer at a ratio of 100 mg tissue/1 ml buffer for 1 min to obtain cardiac tissue extracts. The homogenates were put on ice for 10 min then centrifuged at 12,000 g for 40 min twice. The supernatant was collected and stored at −70 °C.
Electrophoresis and Western Blot
Protein concentration of heart tissue extracts was verified by the Bradford method (Bio-Rad Protein Assay, Hercules, CA, USA). Protein samples (50 μg/lane) were divided on a 10 % SDS polyacrylamide gel electrophoresis (SDS-PAGE) among a fixed voltage of 75 V. Electrophoresed proteins were relocated to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, 0.45 μm pore size) through a transfer equipment (Bio-rad). PVDF membranes were incubated in 5 % milk within TBS buffer. Primary antibodies containing Fas receptor, FADD, Bcl-xL, Bcl-2, Bax, t-Bid, caspase 8, caspase-9, caspase-3, p-Bad, VEGF, p-PI3k (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bad (BD Biosciences, San Jose, California, USA), p-Akt (Cell Signaling Technology, South San Francisco, CA, USA) and α-tubulin (Neo Markers, Fremont, CA, USA) were diluted to 1:500 into antibody binding buffer overnight on 4 °C. The immunoblots were cleaned in TBS buffer three times for 10 min then set in the 2nd antibody solution consisting of donkey anti goat IgG-HRP, goat anti-mouse IgG-HRP or goat anti-rabbit IgG-HRP (Santa Cruz) planned as one hour and diluted five hundred fold in a TBS buffer. The immunoblotted proteins were visualized applying an augmented chemiluminescence ECL western Blotting luminal Reagent (Santa Cruz, CA, USA), also quantified using a Fujifilm LAS-3000 chemiluminescence detection system (Tokyo, Japan).
H&E staining, Masson trichrome staining, and TUNEL
The heart was extracted then soaked in formalin, then dehydrated via graded alcohols, after that embedded in paraffin wax. In heart samples, the 0.2-μm thick paraffin sections were cut from paraffin-embedded tissue blocks. The tissue samples were dewaxed using in xylene immersion, and then rehydrated. For Hematoxylin-eosin staining (H&E staining), the slices were dyed with hematoxylin and eosin. For Masson Trichrome Staining, the slices were dyed with Masson Trichrome. After lightly rinsing with water, the slides were dehydrated through graded alcohols then immersed in Xylene twice. Photomicrographs were obtained by Zeiss Axiophot microscopes. The Terminal Deoxynucleotide Transferase-mediated dUTP Nick End Labeling (TUNEL) assay were studied with an apoptosis detection kit (Roche Applied Science, Indianapolis, IN, USA) and observed that TUNEL-positive nuclei (fragmented DNA) fluoresced bright green at 450–500 nm. The mean number of TUNEL-positive cells were calculated for 5–6 individual fields x 2 slices x 3 regions of the left ventricle (the upper, the middle, the lower) removed from 6 mice hearts within all groups. All data counts were executed in two independent individuals within a blind study.
Statistical analysis
The weight index, protein levels and percentage of TUNEL positive cells were compared with the Control, the Hypoxia, the Hypoxia + RC90 and the Hypoxia + RC270 groups by analysis of variance with pre-planned contrast comparison. In every case P < 0.05 was considered significant.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
M-C L raises the idea of the current studies, participated in the coordination of studies and drafted the manuscript. P-Y P, Y-M L, Y-L Y, S-M C and Y-F L help in animal models, experimental processes and echocardiographic images. M-H L performed the statistical analysis. S-D L, C-Y H and J-G L participated in the study design, funding and coordination. All authors read and approved the final manuscript.