Zhu Xi1, Wang Meng1, Zhao Chengjun, Li Ruosong, Yang Juan1, Yan Guangchang1, Ye Ting2, Zuo Xuezhi2, Liu Liu1, Octavia LS Chong Lee Shin1, Zhu Fengming1, Sun Jie2, Xu Huzi1 ,Zhao Zhi1,Cao Chuyi1,Wang Yuxi1,Yang Qian1,Xu Gang1,Zeng Rui1,Yao Ying1,2 Tongji Medical College, Huazhong University of Science and Technology Tongji Hospital 1Department of Nephrology, 2 Nutrition, China, Hubei, Wuhan, Jiefang Avenue 1095;
3 Wuhan Hege Biotechnology Co., Ltd., No. 630, Hanyang Avenue, Hubei, China
Received 2018.01.20 Received 2018.04.06 Electronic version 2018.06.15 Published 2018.06.30
Abstract: Mucopolysaccharide (GAG), as a component of type II collagen, has a relatively close relationship with bone metabolism. It has been shown that composite collagen can promote the connection of trabecular bone structures. However, the exact mechanism is still unclear. In this paper, we aimed to determine the specific effects and mechanisms of complex collagen on glucocorticoid-induced osteoporosis. GAG was administered daily for 60 days. The results show that the composite collagen has a protective effect on glucocorticoid-induced osteoporosis. After treatment with composite collagen, the number of trabeculae and the density of attachment increased. We generated bone marrow-derived macrophages to explore the effects of composite collagen on osteoclast differentiation. We collected cellular proteins and RNAs present in macrophage cell stimulating factor (M-CSF) and nuclear factor-kB ligand receptor agonist (RANKL) and found that complex collagen inhibits NF-kB and MAPK pathways. Thereby, osteoclast differentiation molecules such as matrix metalloproteinase (MMP 9) and activated T cells, cytoplasmic nuclear factor (NFATc-1) are down-regulated. Our results suggest that composite collagen may have therapeutic potential for the treatment of glucocorticoid-induced osteoporosis in a clinical setting.
Keywords: glucocorticoids, osteoporosis, complex collagen, NF-kB and MAPK
Introduction
Chronic kidney disease (CKD) is characterized by a gradual loss of function of the kidney structure and various causes over a period of months or years, usually greater than 3 months. A study in China shows that 10.8% of Chinese have CKD; that is, the number of CKD patients in China is estimated to be about 119.5 million. Glucocorticoids (GCs) are one of the most commonly used drugs in CKD patients due to their high anti-inflammatory and immunosuppressive effects. However, long-term and high-dose use can re-emerge patients with a large number of adverse complications including glucocorticoid-induced osteoporosis (GIOP).
GIOP is the most common secondary osteoporosis, characterized by osteoporosis, reduced bone strength, and increased risk of fracture. The endogenous and physiological concentrations of GCs play a positive role in the differentiation and activation of osteoblasts and promote the process of physiological bone formation. In contrast, patients treated with GCs experience a very tedious experience. Some patients who are sensitive to GC do not even walk or stand normally after long-term and high-dose GC treatment, so their quality of life is very low. Studies have shown that GCs disrupt the balance of osteoblast, bone cell and osteoclast activity, leading to osteoporosis. A decrease in the number and activity of osteoblasts and bone cells or an increase in the number and activity of osteoclasts alters the balance between bone formation and bone resorption, ultimately leading to osteoporosis.
The NF-kB channel and its downstream molecules, such as MMP 9, CTSK and NFATc-1, are considered to be key regulators closely related to GIOP. Overexpression of these downstream molecules promotes the differentiation of bone marrow macrophages into osteoclasts, leading to osteoporosis.
Type II collagen is a protein mainly expressed in the skin and bones. Collagen fibers, elastin and mucopolysaccharide (GAG), which interweave to form a network of bone density-related structures that increase bone mass and strength. In particular, GAG is used as an important marker for measuring type II collagen and bone metabolism. However, the relevant mechanisms are still unclear.
In this study, we aimed to determine the effect of complex collagen on skeletal processes during osteoclastogenesis. Complex collagen inhibits GC-induced osteoporosis, which promotes osteoclast differentiation by NF-kB and MAPK channels, which promote osteoclast-associated molecules MMP 9, CTSK and NFATc-1. Complex collagen inhibits GCs-mediated osteoclastogenesis, in this way inhibits GC-induced osteoporosis.
Raw materials and methods
Preparation of pure composite collagen solution
The composite collagen solution was prepared by a composite collagen tablet (Wuhan Hege Biotechnology Co., Ltd.). The tablets were dissolved in distilled water at a concentration of 10% (10 g/100 ml); pH was adjusted to 7.0. Finally, the solution is filtered through an anti-bacterial filter to maintain sterility.
Animals and treatment
Male C57bl/6 mice (8 weeks old, weighing 25 g) were purchased from Beijing Huafukang Experimental Animal Technology Co., Ltd. (Beijing, China). At the Animal Care Unit of Tongji Medical College, the animals adapted to the specific living environment under pathogen-free (SPF) conditions. Animals were kept at a temperature of 22 degrees Celsius with a light/dark cycle of 12 h/12 h for 10 weeks until 4.5 months of age. Placebo and prednisolone sustained release pellets were purchased from Innovative Research (IRA, Florida, USA). Animals were randomized into four experimental groups: the normal group (no special intervention), the placebo group (subcutaneously implanted with 7.5 mg of placebo, 60 days of sustained release), and the prednisolone group (to mice) Subcutaneously implanted with 7.5 mg of prednisolone, 60 days of sustained release), GAG group (implanted with the same dose of prednisolone sustained-release group subcutaneously, and given a combined collagen solution); each group 6-8 mice. We chose a pellet with a total dose of 7.5 mg and a release period of 60 days. Mice were anesthetized by injecting 1% sodium pentobarbital (0.008 ml per gram body weight). Then, create a suitable place above the scapula to implant a pellet that slowly releases the placebo and prednisolone. After surgery, the dose of the composite collagen solution was 0.45 mg/day per gram of body weight. After 60 days, the mice were sacrificed by cervical dislocation. We did not find any pellets after 60 days. Cell phone femur, tibia and blood for further analysis. All experimental procedures were conducted in accordance with the guidelines of the National Institutes of Health and approved by the Animal Care and Use Committee of Tongji Hospital (ACUC).
Bone morphology parameter
Bone morphological parameters and microstructural features of the left tibia were analyzed by micro-computed tomography (μ-CT50, Scanco Medical, Barthersdorf, Switzerland). The proximal humerus is reconstructed in three dimensions using built-in software. The trabecular parameters include bone volume/total volume ratio (BV/TV), number of trabeculae (Tb.N), trabecular thickness (Tb.Th), and trabecular space (Tb. Sp), bone area/bone volume ratio (BS/BV), and connectivity density (Conn.D).
Pathological bone coloring
The femur was fixed in a 4% paraformaldehyde solution for 2 days. It was then subjected to decalcification treatment in a 0.5 M ETDA (pH = 8.0) solution for 3 weeks and embedded in paraffin. The paraffin-embedded femur was divided into 5 μm sections, stained with Eosin (H&E), and observed under a microscope (Olympus, Japan).
Immunoblot analysis
Smashing bone tissue in liquid nitrogen and in RIPA lysis buffer (promoting agent, Wuhan, China) containing protease inhibitor mixture (accelerator, Wuhan, China) and phenylmethylsulfonyl fluoride (accelerator, Wuhan, China) ) homogenization. Cellular proteins were collected in the same manner. The total protein concentration (accelerator, Wuhan, China) was determined using a BCA kit according to the manufacturer's instructions. The protein was separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). These membranes were blocked by 5% skim milk in 0.1% to 20 TBS for 1 hour at 37 °C. The OPG antibody was then probed (diluted 1:1000, Bioworld, China), RANKL (diluted 1:1000, ProSci Inc., USA), MMP 9 (diluted 1:1000, Abcam, UK), NFATc-1 (diluted 1 : 1000, Abcam, UK), CTSK (diluted 1:1000; Abcam, UK), total phosphorus-65, -38, -ERK, -JNK (diluted 1:1000, cell signaling technology, USA) and GAPDH (diluted 1 : 4000, Abbkine, China) lasts all night at 4 degrees Celsius. The next day the blot was washed off and incubated with conjugated HRP secondary antibody for 1 hour at 37 °C, visualized by compound light visualization (ECL, BioRad, USA). Analyze the gray value of the target band using ImageJ (NIH, USA).
Cell culture
Primary bone marrow mononuclear cells were isolated from 4 week old male C57bl/6 mice. The femur and tibia were rinsed with the culture medium, and then the bone marrow cells were collected into a disk having a diameter of 10 cm. Cell culture (FBS), 100 U / mL penicillin, 100 U / mL streptomycin and 30 ng / mL of macrophage colony-stimulating factor M-CSF (PeproTech) in α-MEM containing 10% fetal bovine serum , Rocky Hill, New Jersey). After 24 hours, the floating cells were collected in the presence of M-CSF and culture was continued for 3 days. Adherent cells serve as bone marrow-derived macrophages (BMMs) for further use. BMMs are maintained in a humidified incubator (Thermal Science, USA), 37 ° C, 5% CO 2 , 95% atmosphere. The culture is replenished every two days.
Cell activity measurement
Cell count kit-8 (accelerator, Wuhan, China) was used to assess cell viability. BMMs were cultured in each well of a 96-well plate at a density of 5 x 103. After cell adhesion, treatment with different concentrations of composite collagen (GAG: 0, 0.0015, 0.0075, 0.015, 0.15, 1.5 g / L) and dexamethasone (DXM: 0, 0.001, 0.01, 0.1, 1 μM) The cells were 96 hours. The cell culture medium was then replaced with a 10% CCK-8 complete medium. Incubation was carried out at 37 ° C for 1-4 hours, and the absorbance at 450 nm was measured with a multi-mode reader.
TRAP coloring
To detect osteoclast differentiation, BMMs were exposed to 100 ng/mL RANKL (PeproTech, Rocky Hill, NJ) and 30 ng/mL M-CSF. Cells were treated with different concentrations of composite collagen (0, 0.0015, 0.0075, 0.015, 0.15, 1.5 g/L) and dexamethasone (0.1 μM) for 96 hours, then 4% paraformaldehyde according to the manufacturer's instructions. Fixed and stained with tartaric acid phosphatase (TRAP). (Acid phosphatase, white blood cells (TRAP) Kit, Sigma, USA). TRAP positive cells are considered to be osteoclasts and counted.
RNA preparation and reverse transcriptase-polymerase chain reaction
Total ribonucleic acid (Invitrogen, USA) was extracted from osteoclasts using a kit according to the manufacturer's instructions. In a 20 μL reaction system, a microscopic RNA was reverse transcribed into the first strand cDNA by the GoScript reverse transcription system (Promega, USA). The cycle parameters were used as follows: 40 cycles of PF denaturation, heat treatment at 95 degrees for 15 seconds and 60 degrees for 60 seconds. Three samples were repeated for each sample. Quantitative PCR reactions were performed using a fluorescent reagent mix (kit, Germany) under a Swiss Roche 480II lamp. The expression of Mrna expression of osteoclast-associated markers such as MMP 9, CTSK, NFATc-1 and RANKL was determined by comparing the cycle threshold (Ct) method and normalized to GAPDH expression level. The PCR reaction primer sequences are shown in Table 1.
Table 1. Primer sequences
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Statistical Analysis
All data in the current study were reported using the mean ± standard deviation comparison method between the two groups using unpaired or non-parametric Mann-Whitney tests. All data were analyzed using SPSS 18.0 and GraphPad Prism 5.0 software. P < 0.05 was considered to be meaningful.
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Figure 1 Effect of composite collagen on bone microstructure of GIOP mice. The mice were subcutaneously implanted for 60 days with a sustained release of 7.5 mg of placebo pills or hydrogenated Ponisol pills, and the collagen was administered intragastrically.
A: Three-dimensional reconstruction of the proximal humerus under micro-CT analysis from the normal group, placebo group, hydrogenated prednisone group, and composite collagen group.
B: Microct ct analysis of the histogram of the proximal tibia: relative bone volume to total volume ratio (BV/TV); number of trabeculae (Tb.N); trabecular space (Tb.Sp); connection density (Conn. D).
C: Representative image of femur H&E staining in different groups, with a scale of 20 μm. Data are expressed as mean ± standard deviation, n = 5-7 / group. The difference between the treatment group and the control group was statistically significant, and the results showed *P<0.05, **P<0.01.
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Figure 2 Composite collagen inhibits the expression of RANKL/OPG ratio in GIOP mice.
Western blot analysis of RANKL and OPG in A. GIOP mice.
B. The histogram shows that hydrogenated prednisone leads to activation of the RANKL/OPG signaling pathway, thus promoting glucocorticoid-induced osteoporosis (GIOP). Composite collagen reduces the RANKL/OPG ratio in bone tissue, thereby protecting mice from GIOP. All results showed mean ± SD, n = 3-5/group, *P < 0.05.
result
Effect of composite collagen on bone microstructure in GIOP mice
The left tibia bone microstructure was observed by micro-CT. 3D reconstruction techniques were used to directly assess bone structure and bone density. The bone microstructure was calculated by analyzing the trabecular parameters including BV/TV, Tb.N, Tb.Sp and Conn.D. Compared to the normal and placebo groups, the hydrogenated prednisone group showed significant bone loss in the 3D reconstruction (Fig. 1A). Significant drops were observed in the measurement of BV/TV, Tb.N and Conn.D. Compound collagen treatment group, BV/TV, Tb.N and Conn.D.