Echinacoside Suppresses Dexamethasone‑induced Growth Inhibition And Apoptosis in Osteoblastic MC3T3‑E1 Cells
Mar 11, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Abstract.
Glucocorticoids (GCs) are widely used to treat chronic diseases. Prolonged and/or overdose administration of GCs have many side effects on human health including GC-induced osteoporosis (GIOP). In this investigation, the objective was to assess the influence that echinacoside from cistanche (ECH) exerts upon dexamethasone-treated murine osteoblastic MC3T3-E1 cells. We found that ECH(echinacoside from cistanche) (5, 10, 20, and 40 mg/l) inhibited dexamethasone (1,000 nM)-suppressed cell viability as demonstrated by Cell Counting Kit-8 (CCK-8) assay. The dose of 10 mg/l was selected for the following experiments because this dose had a better effect than the dose of 5 mg/l, and the doses >10 mg/l had a similar effect as this dose. ECH(echinacoside from cistanche) (10 mg/l) or pifithrin‑α (PFT-α) (a p53 inhibitor, 20 µM) suppressed dexamethasone-induced MC3T3-E1 apoptosis as illustrated by Annexin V/propidium iodide (PI) double-labeling flow cytometry analysis. ECH(echinacoside from cistanche) or PFT-α treatment also alleviated dexamethasone's action of inhibiting Bcl-2 expression as well as dexamethasone's action of stimulating the expression of p53 and Bax. Moreover, lentivirus mediated-p53 overexpression reversed the effects of ECH(echinacoside from cistanche) in dexamethasone-treated MC3T3-E1 cells, suggesting that ECH(echinacoside from cistanche) induced anti-apoptotic effects in dexamethasone-treated osteoblasts via a p53-dependent pathway. In summary, ECH(echinacoside from cistanche) has a protective effect against osteoblastic cell apoptosis induced by dexamethasone, suggesting that ECH(echinacoside from cistanche) may have the potential for clinical application in the treatment of GIOP.
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Introduction
Glucocorticoids (GCs) are frequently used to treat a wide spectrum of chronic illnesses, including inflammation, cancer, and autoimmune disorders (1). However, prolonged and/or overdose administration of GCs has many side effects, such as hypertension, hyperglycemia, glaucoma, osteonecrosis, and osteoporosis (2-4). It is clear that GC use increases bone loss and fracture risk (3-6). GCs can induce apoptosis of osteoblasts and impair the function of these cells. For instance, dexamethasone, a GC hormone, can upregulate the expression of p53 in osteoblastic MC3T3-E1 cells, thus causing cell apoptosis (7,8). Osteoblast apoptosis leads to decreased bone formation. Oral use of bisphosphonates (alendronate and risedronate) (9,10) has been introduced for the treatment of GC-induced osteoporosis (GIOP). However, low adherence rates limit the efficiency of oral bisphosphonates. Other medications also have limitations such as inconvenient administration [zoledronic acid (11)] and high cost [PTH 1-34 (teriparatide) (12,13)]. Consequently, there is a need for the development of new treatment options for GIOP.
Echinacoside from cistanche (ECH) is a phenylethanoid glycoside isolated from the traditional Chinese medicine Herba Cistanches (14). ECH(echinacoside from cistanche) has protective effects on the neuron system (15-20), liver (21,22), and lung (23) through promoting cell proliferation, and inhibiting inflammatory response, reactive oxygen species (ROS) production, and cell apoptosis. Although ECH(echinacoside from cistanche) can also promote osteoblastic bone regeneration and has extraordinary antiosteoporotic activity in the rat models (24,25), there are no existing records describing the effects of ECH(echinacoside from cistanche) on GC-induced osteoblastic cell apoptosis. Thus, we investigated the influence that ECH(echinacoside from cistanche) exerts upon dexamethasone-induced osteoblastic cell apoptosis and elucidated the preliminary mechanism in MC3T3-E1 cells.
Materials and methods
Cultivation of cells. The Cell Bank of the Chinese Academy of Sciences (Shanghai, China) provided the murine osteoblastic MC3T3-E1 cells. Growth of cells took place in α-minimal essential medium (α-MEM) (Hyclone; GE Healthcare, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Grand Island, NY, USA) and penicillin/streptomycin (Beijing Solar Science and Technology Co., Ltd., Beijing, China). Maintenance of cells was conducted in an environment with 95% air and 5% carbon dioxide at a temperature of 37˚C.
The production of lentivirus overexpressing p53. To create p53 expression construct, the protein-coding sequence (CDS) region of the murine p53 gene was synthesized and inserted into EcoRI/BamHI restriction sites of the lentiviral expression vector pLVX-puro (Clontech Laboratories, Inc., Palo Alto, CA, USA) by Genewiz, Inc. (Beijing, China). The construct was verified by DNA sequencing. 293T cells (Shanghai GeneChem Co., Ltd., Shanghai, China) were transfected with a mixture of plasmids, including viral packaging plasmids and p53 expression plasmid (pLVX-p53) or control plasmid (pLVX) via Lipofectamine 2000 (Invitrogen: Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) based on the manufacturer's instructions. The viral supernatant was collected at 48 h after transfection and used to infect MC3T3-E1 cells. Evaluation of p53 expression was conducted at 48 h after viral infection.
Quantitative polymerase chain reaction (qPCR). We used TRIzol reagent (Invitrogen: Thermo Fisher Scientific, Inc.) to separate total RNA and MC3T3-E1 cells, and RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) according to the manufacturer's instructions to reverse-transcribe total RNA. p53 mRNA level was detected via qPCR on ABI Prism 7300 Sequence Detection System (Applied Biosystems: Thermo Fisher Scientific, Inc., Foster City, CA, USA). The primers were as follows: 5'-CCC CTGTCATCTTTTGTCCCT-3' and 5'-AGCTGGCAGAAT AGCTTATTGAG-3' for p53, 5'-CTGCCCAGAACATCA TCC-3' and 5'-CTCAGATGCCTGCTTCAC-3' for GAPDH.
Then, we assessed p53 mRNA levels via the 2-ΔΔCT method (26) with GAPDH as an internal control.
Western blotting. Lysis of MC3T3-E1 cells took place in radioimmunoprecipitation assay buffer with protease inhibitors (Beijing Solarbio Science & Technology Co., Ltd.), and centrifugation of lysates continued for 15 min at a temperature of 4˚C at 9,600 x g so that precipitation can be removed. Subsequently, we assessed the amount of protein with the BCA assay kit (Thermo Fisher Scientific, Inc.). An equal amount of protein was added onto a 10 or 15% SDS-PAGE and transferred to a nitrocellulose membrane (EMD Millipore, Bradford, MA, USA). The membranes were blocked with 5% skim milk and probed with primary antibodies. After washing 3 times with phosphate-buffered saline (PBS), incubation of membranes with horseradish peroxidase-conjugated secondary antibody (1:1,000; cat no. A0208; Beyotime Institute of Biotechnology, Shanghai, China) was conducted. We conducted western blotting tests via an enhanced chemiluminescence (ECL) kit (EMD Millipore). GAPDH was used as the internal standard. The sources of primary antibodies were as follows: Bax (Sc-493) and Bcl-2 (Sc-492) both from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); p53 (no. 2524) and GAPDH (no. 5174) both from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Experimental grouping. To determine the suitable dose of dexamethasone, treatment of MC3T3-E1 cells was conducted with a series amounts of dexamethasone (0, 1, 10, 100, and 1,000 nM; Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA). At 48 h after incubating cells, Cell Counting Kit-8 (CCK-8) assays were conducted to detect cell proliferation.
To select the appropriate dose of ECH(echinacoside from cistanche), treatment of MC3T3-E1 cells was conducted with 1,000 nM of dexamethasone and various doses of ECH (0, 2.5, 5, 10, 20, and 40 mg/l; Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China). Cells grown under normal conditions (mock) were served as control. CCK-8 assay was performed after 48 h of culture.
For all other experiments, the MC3T3-E1 cells were split into five groups: group 1 (mock), cells were cultured under normal condition; group 2, cells were transduced with pLVX lentivirus; group 3, cells cultured with 10 mg/l of ECH(echinacoside from cistanche); group 4, cells with 20 µM of pifithrin-α (PFT-α; Selleck Chemicals, Houston, TX, USA); group 5, cells transduced with pLVX-p53 lentivirus and treated with 10 mg/l of ECH(echinacoside from cistanche). Cells in groups 2-5 were treated with 1,000 nM dexamethasone. Cell apoptosis and protein expression were measured after 48 h of culture.
Cell proliferation assay. We seeded MC3T3-E1 cells into 96-well plates at 3x103 cells/well and cultured them at normal conditions overnight. Cells were treated as indicated. After 48 h of culture, the CCK8 (Signalway Antibody LLC, College Park, MD, USA) assay was then performed according to the manufacturer's instructions. Briefly, the culture medium in each well was replaced with 100 µl of 10% CCK-8 solution in the medium. After incubating the cells for a duration of 1 h at a temperature of 37˚C, we assessed the absorbance at a wavelength of 450 nm with a microplate reader. The relative cell viability (%) with connections to control wells was calculated.
Cell apoptosis evaluation. We used the Annexin V-FITC cell apoptosis kit (Beyotime Institute of Biotechnology) for quantifying cells that are undergoing early apoptosis following the manufacturer's instructions. In brief, the collection of treated cells was conducted via trypsinization. The cells were washed via PBS and resuspended with Annexin binding buffer. Incubation of cells was conducted via Annexin V-FITC for a duration of 15 min at a temperature of 4˚C followed by incubation with propidium iodide (PI) for 5 min at 4˚C. Cells in the lower right quadrant (Annexin V-FITC positive and PI negative) were considered actively undergoing early apoptosis.
Statistical analysis. Data are the means ± SD for all three independent experiments. Tukey's multiple comparisons test and one-way analysis of variance were conducted to analyze the differences between groups via GraphPad Prism software (version 6.0; GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Establishment of a suitable dose of dexamethasone. To determine the suitable dose of dexamethasone, the MC3T3-E1 cells were exposed to increasing amounts of dexamethasone (0, 1, 10, 100, and 1,000 nM) for 48 h. As shown in Fig. 1, relative cell viability was determined by CCK-8 assay was increased in cells treated with low concentrations of dexamethasone (1 and 10 nM), and significantly reduced in cells exposed to high concentrations of dexamethasone (100 and 1,000 nM).
The most significant reduction was observed in cells with 1,000 nM of dexamethasone. Thus, 1,000 nM of dexamethasone was selected in the subsequent assays.
Effect of ECH(echinacoside from cistanche) on dexamethasone‑induced cell growth inhibition. We then explored the effect of ECH(echinacoside from cistanche) on dexamethasone-induced cell growth inhibition. Treatment of MC3T3-E1 cells was conducted with 1,000 nM of dexamethasone and various doses of ECH(echinacoside from cistanche) (0, 2.5, 5, 10, 20, and 40 mg/l; Fig. 2). At 48 h post-treatment, the addition of ECH(echinacoside from cistanche) at doses of 5, 10, 20, and 40 mg/l notably decreased dexamethasone-induced cell damage. ECH(echinacoside from cistanche) at a dose of 10 mg/l has a more obvious effect than that at a dose of 5 mg/l. Cell viability was similar in cells treated with 10, 20, and 40 mg/l of ECH(echinacoside from cistanche). Therefore, 10 mg/l of ECH(echinacoside from cistanche) was used in the following experiments.
Overexpression of p53 by constructing lentivirus. Dexamethasone can upregulate p53, thereby inducing osteoblasts apoptosis (8). To study the effects of ECH(echinacoside from cistanche) on dexamethasone-induced p53 expression, we constructed a lentivirus encoding p53 (pLVX-p53) and transduced the virus into MC3T3-E1 cells. The expression of p53 was analyzed at 48 h after transduction. Fig. 3 shows that p53 mRNA and protein levels were increased to >2 times in pLVX-p53-transduced cells as compared to cells with control virus (pLVX). p53 was involved in the effect of ECH(echinacoside from cistanche) on dexamethasone-induced apoptosis. The MC3T3-E1 cells were treated with Plavix or pLVX-p53, 10 mg/l of ECH(echinacoside from cistanche), 20 µM of PFT-α (a p53 inhibitor), and 1,000 nM of dexamethasone, and then cell apoptosis was measured via Annexin V-FITC apoptosis kit. As shown in Fig. 4A, dexamethasone treatment remarkably induced cell apoptosis in comparison to the cells cultured under normal conditions (mock). ECH(echinacoside from cistanche) and PFT-α treatment significantly protected the MC3T3‑E1 cells against dexamethasone-induced apoptosis. Additionally, p53 overexpression reversed the protective effects of ECH(echinacoside from cistanche).
Bcl-2 and Bax, as important apoptosis regulators, are known to be regulated by p53 (27). Western blotting indicated that the changes of p53 and Bax protein expression were consistent with the alterations of cell apoptosis, whereas Bcl-2 expression changed in the opposite direction (Fig. 4B and C). Notably, the protein levels of p53 were significantly suppressed by ECH(echinacoside from cistanche) and PFT-α treatment in cells overexpressing p53 and treated with dexamethasone (Fig. 4C). These data suggest that ECH(echinacoside from cistanche) might exert protective effects on dexamethasone-induced apoptosis via inhibiting p53 expression.
Effects of cistanche echinacoside
Discussion
The anti-apoptosis function of ECH(echinacoside from cistanche) has been described in the acute liver injury model induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) and in human neuroblastoma (SH-SY5Y) cells induced by tumor necrosis factor-α or 1-methyl-4-phenylpyridinium ion (MPP+) (17,22,28). It has been reported that dexamethasone treatment can inhibit proliferation and induce MC3T3-E1 cell apoptosis (8,29). In the current study, relative cell viability was significantly reduced in cells exposed to high concentrations of dexamethasone (100 and 1,000 nM), which was consistent with a previous study (8). Additional ECH treatment (5-40 mg/l) remarkably inhibited dexamethasone-suppressed cell viability. Moreover, dexamethasone exposure (1,000 nM) stimulated MC3T3-E1 cell apoptosis, which the ECH treatment restrained to a significant extent. The report gave a first depiction of how ECH is protected against dexamethasone-induced osteoblast cell apoptosis and suggested that ECH may have the potential for clinical application in GIOP.
Furthermore, we tried to explore the preliminary mechanism of the protective effects of ECH(echinacoside from cistanche). Dexamethasone can enhance p53 transcription, thereby inducing osteoblasts' apoptosis and cell cycle arrest (8). p53 is downregulated by ECH in SH-SY5Y cells (28). Here, PFT-α (a p53 inhibitor) had similar protective effects as ECH, while p53 overexpression reversed the protective effects of ECH. The changes of p53 protein expression under different conditions were in agreement with the alterations of cell apoptosis. These results indicate that ECH might exert protective effects on dexamethasone-induced osteoblastic cell apoptosis via inhibiting p53 expression. Bcl-2 family proteins, such as Bcl-2 and Bax, can alter the permeability of the mitochondrial membrane, activate effector caspases, and cause cell apoptosis (30). It is well-known that p53 can regulate Bcl-2 and Bax (27). Here, treatment with PFT-α or ECH(echinacoside from cistanche) attenuated the inhibitory effects of dexamethasone on anti-apoptosis protein Bcl-2 levels as well as the stimulatory effects dexamethasone has on pro-apoptosis protein Bax levels, while p53 overexpression reversed the effects of ECH. These results further demonstrated the involvement of p53 in the protective function of ECH.
In summary, we demonstrated that ECH(echinacoside from cistanche) has a promising protective effect on osteoblastic cell apoptosis induced by dexamethasone, suggesting that ECH may have the potential for clinical application in the treatment of GIOP.
Effects of echinacoside from cistanche
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