Cistanoside A Mediates P38/MAPK Pathway To Inhibit Osteoclast Activity

Abstract

BACKGROUND: Cistanoside A has the effects of anti-inflammation, antioxidation, reducing renal damage, and anti-osteoporosis, but its effect on osteoclast differentiation, function and underlying molecular mechanisms remains unclear. OBJECTIVE: To investigate the effect of Cistanoside A on osteoclast differentiation and bone resorption induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in vitro and its mechanism.

METHODS: Bone marrow macrophages were obtained from the femur and tibia of 4-6-week-old C57BL/6 mice. The cytotoxic effect of Cistanoside A (5, 10, 20, 40, 80, and 160 μmol/L) on bone marrow macrophage viability was examined using the cell counting kit-8 assay kit. Tartrate-resistant acid phosphatase staining was performed to observe the effect of different concentrations of Cistanoside A on osteoblast differentiation and its effective intervention concentration was determined. There was a positive control group, Cistanoside A low, medium, and high dose groups (40, 80, and 160 μmol/L). After cell attachment, 50 ng/mL RANKL was added to induce osteoblast differentiation, and the corresponding dose of Cistanoside A was added to the Cistanoside A low, medium, and high dose groups, respectively. F-actin ring and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride staining were performed to detect the effects of Cistanoside A on the formation of osteoclasts. Toluidine blue staining of bone abrasion slices was used to observe the effects of Cistanoside A on the bone resorption function of osteoclasts. The expression of upstream and downstream proteins of the JNK/MAPK pathway was detected by Western blot. The expression of genes related to osteoclast differentiation and bone resorption function such as tartrate-resistant acid phosphatase, DC-STAMP, Nfatc-1, Ctsk, and c-Fos was detected by RTqPCR. RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay showed that Cistanoside A significantly inhibited RANKL-induced osteoclast differentiation and bone resorption in a dose-dependent manner compared with the positive control group. The results of RT-qPCR showed that compared with the positive control group, both high and low dose groups of Cistanoside A could significantly downregulate the mRNA expression of tartrate-resistant acid phosphatase, DC-STAMP, Nfatc-1, Ctsk and c-Fos in a dose‐dependent manner. The results of western blot assay showed that the high dose group of Cistanoside A significantly inhibited the expression of p-JNK protein at 10, 20, 30 and 60 minutes of intervention; compared with the positive control group, Cistanoside A significantly inhibited the expression of Nfatc1 and c-Fos proteins in a dose-dependent manner. To conclude, Cistanoside A could inhibit the formation and bone resorption of osteoclasts by reducing the level of p-JNK protein, inhibiting the activation of MAPK pathway and the expression of key genes in osteoclasts. 

Keywords: Cistanoside A; osteoclast; MAPK; JNK; RANKL; RANK; osteoporosis; phenylethanoid glycosides 

CISTANCHE TO WORK TO  INHIBIT OSTEOCLAST ACTIVITY

0 Introduction Introduction

Epidemiological studies have shown that as the global population grows and ages, the incidence of osteolytic diseases such as osteoporosis and osteoarthritis will further increase [1]. Current research shows that in the pathogenesis of osteolytic diseases, the imbalance of bone resorption and bone reconstruction caused by excessive activation of osteoclast function will lead to a significant reduction in bone volume, bone density and bone microstructure. Harmful deterioration, leads to increased bone fragility and increased risk of fractures [2-3]. Therefore, osteoclasts are a major cellular target for the identification and development of effective agents to treat osteoporosis. However, currently commonly used drugs that inhibit osteoclasts such as denosumab (anti-RANKL antibody), bisphosphonates, etc. have adverse reactions such as nephrotoxicity, osteonecrosis, risk of endometrial cancer, and osteonecrosis of the jaw [4-5 ]. Therefore, it is of high clinical significance to find new drugs that can inhibit osteoclast activity.

The differentiation and bone resorption function of osteoclasts are mainly regulated by two signaling molecules, macrophage colony-stimulating factor and receptor activator of nuclear factor κB ligand (RANKL) [6]. The colony-stimulating factor 1 receptor (c-fms) on the osteoclast precursor cell membrane binds to macrophage colony-stimulating factor and promotes the expression of receptor activator of nuclear factor kappa B (RANK). RANKL and RANK binding on the cell surface activates transcription factors such as nuclear factor κB, JNK, p38, and JNK, which in turn activates the transcription factor c-Fos, nuclear factor of activated T cells c1 (Nfatc1), etc. in the nucleus, activating the destruction of Bone precursor cells form multinucleated cells and eventually differentiate into osteoclasts [7-9].

Cistanche deserticola is one of the most commonly used Chinese medicinal materials in clinical practice. It is the dried fleshy stem with scaly leaves of Cistanche deserticola or Cistanche tuberosum of the Orobanchiaceae plant [10]. Traditional medicine believes that Cistanche deserticola is warm in nature, sweet and salty in taste, and returns to the kidney and large intestine meridians. It has the effects of nourishing kidney yang, replenishing essence and blood, moisturizing the intestines and laxative, and is used to treat impotence, infertility, muscles and bones caused by insufficient kidney yang and essence and blood deficiency. Diseases such as weakness are in line with the liver and kidney tonic treatment methods used in traditional medicine to treat osteolytic diseases. Pharmacological studies have shown that the main active ingredient of Cistanche deserticola is phenylethanoid glycosides, which has good safety and extensive medicinal functions [11]. Cistanoside A (Molecular formula: C36H48O20) is a natural phenylethanol glycoside compound isolated and purified from the traditional Chinese medicine Cistanche deserticola. It has anti-inflammatory, antioxidant and anti-tumor effects [12-14]. Studies have shown that Cistanche deserticola A can reduce c-Fos levels in primary cells of mice with ethanol-induced acute liver injury [15]. Other studies have shown that Cistanche A can improve trabecular bone structural parameters in ovariectomized osteoporotic mice through the RANKL/TNF receptor-associated factor 6 (TRAF6) pathway [16]. However, there is a lack of further research on the effect of Cistancheside A on osteoclast differentiation and potential molecular mechanisms. This study aims to explore the effect of Cistancheside A on osteoclast differentiation and bone resorption function in vitro and the potential molecular mechanisms, in order to find out New potential drugs to inhibit osteoclastic differentiation.

1 Materials and methods Materials and methods

1.1 Design

For in vitro cell experiments, t-test and one-way ANOVA were performed.

1.2 Time and place

Completed from December 2022 to December 2023 in the Bone and Soft Tissue Injury Repair Laboratory of the Second Hospital of Shanxi Medical University.

1.3 Materials

1.3.1 Experimental animals

  SPF grade 4-week-old C57BL/6 mice, male or female, were purchased from the Experimental Animal Center of Shanxi Medical University, production license number: SCXK (Jin) 2019-0004. Experimental mice were kept in a standard environment: room temperature 26°C, humidity about 55%, light exposure for 12 hours a day, free movement, and eating independently. The experimental protocol was approved by the Experimental Animal Welfare and Ethics Committee of the Second Hospital of Shanxi Medical University (Approval No.: DW2023031).

1.3.2 Reagents and drugs

Cistancheside A (purity 98%, CAS 93236-42-1) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; fetal bovine serum and high-glucose αMEM culture medium were purchased from HyClone Company of the United States; trypsin-EDTA digestion solution 0.25%, Red blood cell lysis solution, 1×PBS, penicillin-streptomycin mixture (×100), toluidine blue staining solution (1/100, borate method), rhodamine-labeled phalloidin ring

Peptide (Cat. No. CA1610) and DAPI stain (ready-to-use) were purchased from Beijing Solebao Technology Co., Ltd., and mouse recombinant macrophage colony-stimulating factor and mouse recombinant tumor necrosis factor ligand superfamily RANKL were purchased from Beijing Ze Xiyuan Biotechnology Co., Ltd.; CCK-8 kit was purchased from MCE Company of the United States, tartarte resistant acid phosphatase (TRAP) staining kit was purchased from Sigma-Aldrich Company of the United States, RNAiso Plus, Premix type reverse transcription Reagents, TB Green® Fast qPCR Mix,

Prime Script TM RT Master Mix and Trizol were purchased from Takara Company in the United States; dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, CTSK, c-Fos, NFATc-1 specific PCR Primers were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; β-actin antibody, c-Fos antibody, Nfatc1 antibody, JNK antibody and p-JNK antibody, m-IgG Fc BP-HRP were purchased from Santa Cruz, USA.

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1.4.6 Bone resorption experiment to test the bone resorption capacity of osteoclasts

Bone marrow macrophages were seeded into a 96-well plate with sterilized bovine bone slices placed in advance at a density of 7×103/well. 100 μL of culture medium was added to each well and divided into positive control group and Cistanche A low-dose. group (40 μmol/L) and Cistancheside A high-dose group (160 μmol/L), each group was equipped with 5 multiple holes, of which 3 multiple holes were placed with bovine bone slices, and the other 2 multiple holes were used to observe the expression of bone marrow macrophages. Adhesive and osteoclast formation. After the cells adhered, the positive control group and cistanches

RANKL at 50 ng/mL was added to all groups to induce osteoclast differentiation. Cistancheside A low- and high-dose groups were added with 40 μmol/L or 160 μmol/L Cistancheside A respectively for intervention, and the medium was changed every 48 h. After culturing for 14 days, take out the bovine bone slices, wash the bone slices twice with PBS, add 70% isopropyl alcohol and high-power ultrasonic cleaning for 15 minutes to wash away the remaining cells, add toluidine blue (1%, dissolved in water) dye solution, and stain at room temperature for 2 seconds. After 1 min, rinse the bone fragments with double-distilled water to remove residual dye. The stained bone slices were observed and photographed using a stereomicroscope, and the area of bone resorption lacunae was counted using Image J software.

1.4.7 RT-PCR

  To detect the intracellular bone resorption function and the expression of osteoclast-related genes, bone marrow macrophages were seeded in a 6-well plate at 1.5×105/well, and 100 μL of culture medium was added to each well, and divided into positive control group, Cistanche A low-dose group (40 μmol/L) and Cistancheside A high-dose group (160 μmol/L). Both the positive control group and the Cistancheside A group were added with 50 ng/mL RANKL inducer.

To induce osteoclast differentiation, 40 μmol/L or 160 μmol/L of Cistancheside A was added to the low- and high-dose groups of Cistancheside A respectively for intervention. The medium was changed every 48 hours. Observation was carried out under an inverted microscope three or four days after induction. It was observed under the microscope that bone marrow macrophages had just fused. Add TRIzol to lyse cells, add chloroform, place on ice for 10 minutes, and aspirate the mixture

In 1.5 mL EP tubes, add 200 μL chloroform to each tube, shake for 15 s, then let stand on ice for 15 min, centrifuge at 4°C, 12 000 r/min for 10 min, take the supernatant, add an equal amount of isopropyl alcohol, and place on ice. Let stand for 10 minutes, discard the supernatant, centrifuge at 4°C at 120,000 r/min, wash with 75% ethanol solution with a volume fraction of sterile enzyme-free water, and dry in the air to obtain total RNA. Use the Prime ScriptTMRT Master mix reverse transcription kit to configure a 20 μL system for reverse transcription to synthesize cDNA. Then configure the PCR working system according to the kit instructions and perform detection in a fluorescence quantitative PCR instrument. PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, a total of 40 cycles. The primer sequence is shown in Table 1.

After adding simple α-MEN medium and starving for 3 h, the Cistancheside A group was also added with 160 μmol/L Cistancheside A for intervention. After the end of starvation, 100 ng/mL RANKL was added to each group for stimulation. The stimulation times were 0, 10, 20, 30, and 60 min respectively. After the stimulation time was completed, the culture medium was discarded and washed three times with pre-cooled 1×PBS. Extracted proteins were used to detect the expression of p-JNK, JNK and β-actin proteins.

After the protein extraction is completed, use gel electrophoresis to separate the protein and transfer it to a PVDF membrane. Add 5% bovine serum albumin (BSA) for blocking at 4°C for 1 hour. Discard the blocking solution, wash the membrane 3 times with TBST buffer, and add primary antibody. (Nfatc1 antibody 1: 1000, c-Fos antibody 1: 1000, p-JNK antibody 1: 1000, JNK antibody 1: 1000, β-actin antibody 1: 1000), incubate at 4°C for 14-16 h , wash the membrane 3 times with TBST buffer, add secondary antibody (m-IgG Fc BP-HRP, dilution ratio 1:5000), incubate at room temperature for 2 hours, add fluorescent solution after washing, and develop under the gel imaging system, Image Lab The software analyzes the gray value of the obtained bands, and uses β-actin as the internal reference to calculate the relative expression of the target protein.

1.5 Main observation indicators

  ① The results of RANKL-induced osteoclast differentiation after Cistancheside A intervention; ② The formation of actin rings on osteoclast surface and changes in osteoclast bone resorption function after Cistancheside A intervention; ③ The key to osteoclasts after Cistancheside A intervention Gene expression; ④ Expression of downstream proteins of the JNK/mitogen-activated protein kinase (MAPK) pathway and p-JNK protein after Cistancheside A intervention.

1.6 Statistical analysis

GraphPad Prism 9 software was used for statistical analysis and drawing. All data were expressed as x-±s. t test and one-way analysis of variance were selected for comparison between groups. P < 0.05 was defined as a significant difference. The statistical methods of this article have been developed by the Statistics Department of Shanxi Medical University.

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