Acteoside Derived From Cistanche Improves Glucocorticoid-Induced Osteoporosis By Activating PI3K/AKT/mTOR Pathway

Abstract

Objective

Glucocorticoids are widely used in clinical practice; however, they can cause side effects, such as osteoporosis. Acteoside (ACT) from Cistanche has been used to combat a variety of diseases. The study was conducted to evaluate the efficacy of ACT in glucocorticoid-induced osteoporosis (GIOP) and its potential mechanism.

Methods
Dexamethasone (Dex) was injected intramuscularly to induce osteoporosis in a rat model, and ACT was given orally. ACT was supplemented in vivo in Dex-stimulated osteoblastic MC3T3-E1 cells. RT-qPCR was performed to assess the mRNA levels of bone formation (Runx2, CoL1A1), and bone resorption (OPG and RANKL). A commercial ELISA kit was applied to assess serum OC and CTX levels. Western blot was performed to assess protein levels in the PI3K/AKT/mTOR signaling pathway. CCK-8 assay and flow cytometry were performed to assess osteoblast viability and apoptosis.

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Results
ACT reduced Dex-induced bone microstructure deterioration, increased serum levels of OC, and decreased the levels of CTX (P < 0.05). In the MC3T3-E1 cells, Dex inhibited cell viability and promoted apoptosis; however, this effect was greatly attenuated by ACT (P < 0.05). Concurrently, ACT reversed the reduction in Runx2, osterix, CoL1A1, and OPG mRNA levels, ALP activity, and the promotion of RANKL by Dex. Additionally, ACT attenuated Dex-induced inhibition of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K protein levels by Dex (P < 0.05), while the PI3K/AKT/mTOR pathway inhibitor LY294002 diminished the potential effect of ACT (P < 0.05).

Conclusion
ACT from Cistanche may exert osteoprotective effects by activating the PI3K/AKT/mTOR signaling pathway to alleviate Dex-induced osteoporosis.

Keywords: Acteoside Cistanche glucocorticoids-induced osteoporosis

Introduction
Osteoporosis (OP) is a generalized skeletal disease characterized by low bone mass, destruction of bone tissue microstructure, and imbalance in bone homeostasis [1]. OP causes more than 8.9 million fractures per year and osteoporotic fractures occur almost every 3 seconds, and leading to lifelong disability or death [2]. Glucocorticoids (GCs) are commonly administered for the treatment of noninfectious inflammatory diseases (such as asthma and inflammatory bowel disease) as well as autoimmune conditions [3]. However, long-term GC use predisposes to more than 30% of OP cases and more than 10% of osteonecrosis cases [4]. GCs directly inhibit osteoblast proliferation and differentiation [5], diminish maturation and activity, and induce apoptosis of osteoblasts, which in turn leads to the development of glucocorticoid-induced osteoporosis (GIOP) [6]. Moreover, growing evidence suggests that osteoblast dysfunction is the major cause of bone loss. Therefore, an increase in the number and function of osteoblasts appears to be the main strategy to prevent of OP, especially GIOP.

Herbal medicines, especially those based on natural compounds, have been practiced for thousands of years for the treatment of various diseases [7]. Cistanche is a precious herb recorded in the Chinese Pharmacopeia that grows in the southern area of the Xinjiang Uygur Autonomous Region and is known as desert ginseng [8]. Cistanche has been proven to have various effects, such as immune enhancement, renal protection, anti-ageing, antioxidant, anti-inflammatory, and neuroprotective effects [9]. Acteoside (ACT) is a phenyl ethanolic glucoside in Cistanche, which has biological activities, such as inhibition of neuronal apoptosis [10], alleviation of liver damage [11], anti-inflammation [12], antioxidation [13], prevention of diabetes [14], and obesity [15], as well as the degeneration of the articular cartilage [16]. Furthermore, several studies have reported the pharmacokinetics of ACT and confirmed that the therapeutic effect exists even if the oral utilization is only 0.12%. ACT is considered a prodrug with active metabolites in vivo [17].

Zhang et al. demonstrated a potential protective effect of ACT on ovariectomy-induced bone loss in rats [18]. ACT modulates the IGF-1/PI3K/mTOR signaling pathway to prevent bone loss in diabetic rats [19]. The PI3K/AKT/mTOR pathway has been reported to have critical regulatory roles in various diseases, including GIOP [20]. Naringin alleviates GIOP by regulating the PI3K/AKT/mTOR signaling pathway [21]. Additionally, this signaling pathway regulates osteoblast differentiation [22], bone formation [23], and fracture healing [24]. However, the potential role of ACT on GIOP and whether it is regulated by the modulation of the PI3K/AKT/mTOR signaling pathway is unknown.

The present study aimed to understand the protective effects of ACT in a GIOP rat model and osteoblasts in vitro and to elucidate the underlying molecular mechanisms.

Materials and methods
Experimental animals
Forty 8-week-old male Sprague-Dawley (SD) rats weighing 280–340 g were obtained from Shanghai Laboratory Animal Center (CAS, Shanghai, China). The care procedures of the experimental animals were carried out following the protocol approved by The Third Affiliated Hospital of Qiqihar Medical University Animal Ethics Committee. And the study was conducted in agreement with the ARRIVE guidelines. Animals were housed in animal centers free of pathogens, 24 ± 2 °C, 12 h light and dark cycles, 50 ± 5% humidity, and with free access to food and water.

GIOP model construction
After 7 days of adaptive feeding, rats were randomly divided into control and model groups using the random number table method, Dexamethasone (Dex, Sigma-Aldrich St. Louis, MO, USA) 5 mg/kg twice a week for 6 weeks was injected intramuscularly to induce OP in rats in the model group [25]. Rats in the control group (n = 10) were injected with an equal amount of saline. No significant changes in body weight were observed in the rats in both groups at 6 weeks. Subsequently, the rats in the model group were subdivided into the model group, ACT low dose group (Model + ACT-L) group, and ACT high dose group (Model + ACT-H) group. ACT (HPLC 98%) was obtained from Baoji Herbest Bio-tech., Ltd. (Baoji, China), and treated with an aqueous solvent suspension. A dose of 50 mg/kg/d and 100 mg/kg/d ACT was administered orally to rats in the low and high dose groups (8 rats per group), respectively, and was continued for 8 weeks. Among them, the sample size was calculated based on a previous study [26]. Considering a two-sided significance level of 0.05 [95% confidence interval (α = 0.05)], 80% power, and an effect size of 0.5, power analysis estimated a needed sample size of 6 animals per group assuming a relevant effect level of 30% osteoporosis. Further, with a dropout rate of 20%, 8 animals per group (total of 32 rats) were considered ideal.

Rat serum samples
After 8 weeks, rats were anesthetized with 10% chloral hydrate (400 mg/kg, intraperitoneally, Solarbio, Beijing China), blood was collected from the abdominal aorta, clotted at room temperature, and centrifuged at 3500 rpm/min for 10 min. The serum was stored in 1.5 mL centrifuge tubes until further use.

Bone mineral density assessment
Bone mineral density (BMD) was using by a DAX small animal bone densitometer (Ranzhe Instrument Equipment Co., Shanghai, China). Briefly, the rats were placed in the supine position and the hind limbs were positioned outward. The right femur of the rats was scanned according to the manufacturer’s instructions, and the BMD values were recorded [27].

Bone morphology analysis
At the end of the experiment, the rats were euthanized by intraperitoneal injection of excess chloral hydrate, and the femurs were fixed in 70% ethanol. Subsequently, scans were performed on a VivaCT 40μCT system as described in previous study and the bone volume/total volume (BV/TV), bone trabecular number (Tb.N), bone trabecular thickness (Tb.Tn), and bone trabecular separation (Tb.Sp) were assessed [28]. For euthanasia, SD rats were anesthetized after the bone morphology analysis by an intraperitoneal injection of 10% chloral hydrate (350 mg/kg).

Enzyme-linked immunosorbent assay (ELISA)
Commercial ELISA kits were used for the detection of osteocalcin (OC, E-EL-R0243c, Elabscience) and C-terminal telopeptide (CTX, E-EL-R1405c, Elabscience) levels in the serum of rats. Briefly, the reagents were removed at 18-25 °C to equilibrate and prepare the solution for washing and standard working. Enzyme plates Plates were set up with standard, blank, and sample wells. About 100 μL sample diluent solution was added to the wells, incubated for 90 min at 37 °C, and replaced with a biotinylated antibody for 60 min at 37 °C. After washing, the enzyme-binding working solution was added and incubated for 30 min. The substrate solution (90 μL) was added, and incubated for 15 min with protection from light. When the blue gradient appeared in the standard wells, 50 μL of the termination solution was added and the change in OD450 was analyzed.

Reverse transcription-quantitative real-time PCR (RT-qPCR)
TRlzol LS reagent was added to the serum of rats and ACT-treated MC3T3-E1 cells, and total RNA was extracted by incubation at room temperature followed by the addition of chloroform. RNA concentration and purity were confirmed by measuring A260/280 absorbance in a Nanodrop ND-1000 spectrophotometer. The SuperScript II Reverse Transcriptase kit was used for the reverse transcription of RNA to cDNA. Subsequently, cDNA was applied as a template, a primer was added, and the SYBR-Green PCR master mix kit was mixed and supplemented with ddH2O to 20 μL, and the PCR amplification reaction was performed using the Applied biosystems AMI7500 Fast Real-time PCR system (ABI, CA, USA). GAPDH was used as an internal reference for normalization, and the relative expression levels were calculated using 2−ΔΔCt method and performed in triplicates. The primer sequence for Runt-related transcription factor 2 (Runx2), collagen type I α1 (CoL1A1), receptor activator of nuclear factor-κB ligand (RANKL), osterix, and osteoprotegerin (OPG) are presented in Table 1.

Western-blot analysis
RIPA lysate containing 1% protease inhibitor was added to the cells, and total protein from each group was collected after lysis of the cells by shaking for 5 min at 4 °C. BCA assay kit was employed to quantify the concentration of proteins in each group. Following this, the protein samples were mixed with 10% sodium dodecyl sulfate (SDS) buffer in equal volume and boiled in a water bath at 95 °C for 5 min for protein denaturation. A 12% SDS-polyacrylamide vertical gel was prepared, and after the gel solidified, 30 μg of protein was sampled for the 120 mA 90 min gel electrophoresis separation. The protein was then transferred onto the PVDF membrane at 90 V for 120 min. After sealing with 5% bovine serum albumin, the PVDF membrane was cut based on the molecular weight of the marker and target protein band and incubated with the corresponding primary antibody overnight at 4 °C. Rabbit polyclonal antibodies against p-AKT, p-mTOR, and p-PI3K were diluted to ratio of 1:1000 (Proteintech, Wuhan, China). TBST was washed for 30 min and incubated with the corresponding horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. The blot was visualized using enhanced chemiluminescence reagents. The density of protein bands was quantified using Image J. GAPDH was employed as a control to calculate relative protein levels.

Cell culture and grouping
Mouse pre-osteoblasts MC3T3-E1 were purchased from the Chinese Tissue Culture Collections (CTCC, China) and cultured in α-MEM containing 10% fetal bovine serum and 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator at 37 °C and 5% CO2. For bone differentiation culture, osteogenic induction medium was made by mixing 10 mM-β-glycerophosphate and 50 mg/mL of ascorbic acid (Sigma-Aldrich) with α-MEM and added to the MC3T3-E1 cells to induce their differentiation. The medium was changed after 3 days. Cultures for 14 days were used for Alkaline phosphatase (ALP) viability. Furthermore, cells were pretreated with the PI3K inhibitor LY294002 (10 μM) for 30 min, followed by treatment with Dex and ACT.

Cell viability assay
The Cell Counting Kit-8 (CCK-8) assay was performed to assess the viability of osteoblasts treated with ACT. The MC3T3-E1 cells were inoculated into 96 well plates at 5 × 103 cells/well. A quantity of 1 μM of Dex was considered based on previous studies [29] and co-incubated with gradient concentrations of ACT (10−8, 10−7, 10−6, and 10−5 M). The culture medium in the 96-well plates was replaced after adding a mixing medium and CCK-8 reagent in a 10: 1 ratio. After continued incubation for 1 h at 37 °C, the change in OD450 was assessed.

Osteoblast apoptosis number detection
The MC3T3-E1 cells that were subjected to different treatments were digested using EDTA-free trypsin and collected by centrifugation. After washing with a 1 × binding buffer, 5 μL Annexin V-FITC was added and incubated at room temperature for 5 min, supplemented with 5 μL PI and passed through a 200-mesh nylon net. The samples were analyzed using FACScan flow cytometry system.

ALP activity assay
The MC3T3-E1 cells were inoculated into 24-well plates, and ACT and Dex were added after plastering. The cell culture medium was replaced with an osteogenic differentiation medium for culture. After using lysis of cells by cell lysis solution (P0012J, Beyotime Biotechnology), the supernatant was collected by centrifugation, and the standard curve was plotted. An ALP kit (P0132S, Beyotime Biotechnology) was used to evaluate ALP activity and the OD450 was calculated.

Statistical analysis
After performing three independent experiments in triplicate, the data were analyzed using GraphPad Prism 6.0 software and expressed as mean ± standard deviation. ANOVA was employed to compare statistical differences between multiple groups. P-value < 0.05 was considered statistically different.