Volume 27, Issue 4 (6-2020)
RJMS 2020, 27(4): 49-59 |
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Research code: 20821423981007
Ethics code: IR.IAU.REC.1398.32
Clinical trials code: IR.IAU.REC.1398.32
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Biniaz B, abbaszadeh H, farzanegi P. Effect of Stem Cells and Glucosamine on expression of BSPII gene and CPII levels in cartilage tissue of arthritis rats exposed to weight bearing exercise. RJMS 2020; 27 (4) :49-59
URL: http://rjms.iums.ac.ir/article-1-6251-en.html
URL: http://rjms.iums.ac.ir/article-1-6251-en.html
Islamic Azad University, Sari, Iran , h.abaszade61@gmail.com
Abstract: (2052 Views)
Background: Osteoarthritis is the result of a defect in a movable joint covered by a synovial membrane and is characterized by degeneration of cartilage cartilage (1). Concentrations of specific molecules in cartilage and bone that reflect tissue regeneration in the group of patients with osteoarthritis are considered as prognostic factors for joint destruction (2). Among these, proteins such as BSP can be one of these factors. This bone protein stimulates the formation of hydroxyapatite in vitro. BSP binds to hydroxyapatite and contains an RGD integrin binding sequence. It may therefore serve as an adhesive molecule in the cell that allows cells to attach to the extracellular matrix. Another important protein is propeptide C of collagen type (CPII) II. The rate of type II collagen synthesis is directly proportional to the content of CPII in cartilage (3). The present study was performed to investigate the effect of stem cells and glucosamine on BSPII gene expression and CPII levels in cartilage tissue of arthritic rats exposed to weight bearing training.
Methods: This was an experimental study. For this purpose, the number of 70 male wistar rats were randomly divided into two groups: Control-healthy, control-patient, exercise-patient, cell-patient, glucosamine-patient, cell-glucosamine-patient, exercise-glucosamine-patient, cell-exercise-patient, saline-patient, and cell-glucosamine-exercise-patient. Osteoarthritis was induced in male mice with damage to the menisci and cartilage. The training program consisted of 30 minutes of running on a sloping trampoline at a speed of 16 meters per minute. Bone-derived stem cells were also injected into mice at the rate of 1,000,000 cells per kilogram. Glucamine was given orally to mice at a dose of 250 mg / kg / day. After opening the abdominal cavity, samples of cartilage tissue were taken and frozen at -80 ° C for chemical analysis. CPII tissue levels were assessed by ELISA according to the manufacturer's instructions (IBEX Montreal, Quebec, Canada). To measure the expression of BSPII gene in cartilage tissue, cartilage samples were homogenized in phosphate buffer (pH 7.0) at 4° C with a homogenizer. Total RNAs were extracted from the cartilage tissue of all mice using the RNX-Plus kit (SinaClon; RN7713C). The ND-1000 Nanodrop spectrophotometer (Thermo Sci., Newington NH) was used to estimate the quantity and quality of the extracted RNAs. The expression of BSPII gene was performed by Real-Time PCR method and the mRNA levels were normalized to the amount of GAPDH mRNA as the reference gene.The amount of ∆Ct was calculated using the formula [∆Ct = CTBSPII-CTGAPDH]. Then, the gene of expression values were analyzed using the 2-∆Ct formula.To evaluate the significant changes in each of the research variables, between the different groups, one-way analysis of variance and if a statistically significant difference is observed from Tukey post hoc test was used to determine the location of intergroup differences, significance level was considered for all calculations p<0.05, all statistical operations were performed using GraghPadprism 8 software.
Results: The results of analysis of variance for expression of BSPII gene and CPII tissue levels of different groups are shown in Table 1. The calculated F value (60.20, 85.15, respectively) and its significance at the level of p<0.0001, indicate a significant difference between BSPII gene expression and CPII tissue levels between different research groups (Table 1). The highest decrease in BSPII gene expression and CPII tissue levels was observed in the exercise-glucosamine-cell-patient group and the highest increase was observed in the control-patient group (Figs 1 and 2).
Table 1. ANOVA test results for BSPII gene expression and CPII surfaces in the cartilage tissue
* Significant differences
Fig. 1. Comparison of mean BSP gene expression in different research groups.
Normal: Control-healthy, Patient: Control-patient, saline: Saline-patient, Exe: Exercise-patient, MSc: Cell-patient, Glu: Glucosamine-patient, MSc.Glu: Cell-glucosamine-patient, Glu.Exe: Patient-Glucosamine-Exercise, Exe.MSc: Patient-Exercise-Cell, Exe.MSc.Glu: Glucosamine-Exercise-Patient.a: Significance relative to healthy control, b: Significance relative to patient, c: Mean Glucamamine, d: significant relative to cell group, e: significant relative to exercise group.
Fig. 2. Comparison of mean CPII levels in different research groups.
Normal: Control-healthy, Patient: Control-patient, saline: Saline-patient, Exe: Exercise-patient, MSc: Cell-patient, Glu: Glucosamine-patient, MSc.Glu: Cell-glucosamine-patient, Glu.Exe: Patient-Glucosamine-Exercise, Exe.MSc: Patient-Exercise-Cell, Exe.MSc.Glu: Glucosamine-Exercise-Patient.a: Significance relative to healthy control, b: Significance relative to patient, c: Mean You have relative to glucosamine, d: significant relative to cell group.
Conclusion: The results of the present study showed that the expression of BSPII gene and CPII levels of cartilage tissue in the model of osteoarthritis mice increased abnormally. BSP is a bone-specific protein produced by osteoblasts. BSP is specifically enriched between the cartilage-bone phases (3). Propeptide C of type II collagen (CPII) also acts as procollagen by the activity of a proteinase C and is released to form fibrils. The peptide is released from the cartilage and enters the bloodstream. Therefore, its presence in body fluids is one of the indicators of collagen synthesis in cartilage (3). In the present study, CPII levels and BSPII gene expression were assessed at 8 weeks using a osteoarthritis model and the protective effect of exercise, glucosamine and stem cells. The results of our study showed that glucosamine consumption, stem cell injection, exercise or a combination of them effectively control cartilage depletion in a model of osteoarthritis mice. CPII levels and BSPII gene expression were reduced to normal by combined exercise + cell + glucosamine intervention. These values had the most significant decrease in the combined group compared to the osteoarthritis group. These results suggest that the use of exercise in combination with glucosamine intake and cell injection has a synergistic effect on cartilage protection. In the mouse osteoarthritis model, glucosamine inhibits type II collagen degradation by preserving proteoglycans and enhances type II collagen synthesis in articular cartilage. In addition, glucosamine in the early stages of experimental osteoarthritis has a positive effect on the regeneration, structure and mineralization of bone under cartilage and can increase the production of hyaluronic acid in synovial tissue (16). Overall, the findings showed that osteoarthritis increased the expression of BSPII gene and CPII levels in cartilage tissue and a combination of cell + glucosamine + exercise was more effective than exercise, cell and glucosamine alone to reduce these markers.
Methods: This was an experimental study. For this purpose, the number of 70 male wistar rats were randomly divided into two groups: Control-healthy, control-patient, exercise-patient, cell-patient, glucosamine-patient, cell-glucosamine-patient, exercise-glucosamine-patient, cell-exercise-patient, saline-patient, and cell-glucosamine-exercise-patient. Osteoarthritis was induced in male mice with damage to the menisci and cartilage. The training program consisted of 30 minutes of running on a sloping trampoline at a speed of 16 meters per minute. Bone-derived stem cells were also injected into mice at the rate of 1,000,000 cells per kilogram. Glucamine was given orally to mice at a dose of 250 mg / kg / day. After opening the abdominal cavity, samples of cartilage tissue were taken and frozen at -80 ° C for chemical analysis. CPII tissue levels were assessed by ELISA according to the manufacturer's instructions (IBEX Montreal, Quebec, Canada). To measure the expression of BSPII gene in cartilage tissue, cartilage samples were homogenized in phosphate buffer (pH 7.0) at 4° C with a homogenizer. Total RNAs were extracted from the cartilage tissue of all mice using the RNX-Plus kit (SinaClon; RN7713C). The ND-1000 Nanodrop spectrophotometer (Thermo Sci., Newington NH) was used to estimate the quantity and quality of the extracted RNAs. The expression of BSPII gene was performed by Real-Time PCR method and the mRNA levels were normalized to the amount of GAPDH mRNA as the reference gene.The amount of ∆Ct was calculated using the formula [∆Ct = CTBSPII-CTGAPDH]. Then, the gene of expression values were analyzed using the 2-∆Ct formula.To evaluate the significant changes in each of the research variables, between the different groups, one-way analysis of variance and if a statistically significant difference is observed from Tukey post hoc test was used to determine the location of intergroup differences, significance level was considered for all calculations p<0.05, all statistical operations were performed using GraghPadprism 8 software.
Results: The results of analysis of variance for expression of BSPII gene and CPII tissue levels of different groups are shown in Table 1. The calculated F value (60.20, 85.15, respectively) and its significance at the level of p<0.0001, indicate a significant difference between BSPII gene expression and CPII tissue levels between different research groups (Table 1). The highest decrease in BSPII gene expression and CPII tissue levels was observed in the exercise-glucosamine-cell-patient group and the highest increase was observed in the control-patient group (Figs 1 and 2).
Table 1. ANOVA test results for BSPII gene expression and CPII surfaces in the cartilage tissue
| P | F | variable |
| * p <0.0001 | 60.20 | expression of BSPII gene |
| * p <0.0001 | 85.15 | CPII tissue levels |
Fig. 1. Comparison of mean BSP gene expression in different research groups.
Normal: Control-healthy, Patient: Control-patient, saline: Saline-patient, Exe: Exercise-patient, MSc: Cell-patient, Glu: Glucosamine-patient, MSc.Glu: Cell-glucosamine-patient, Glu.Exe: Patient-Glucosamine-Exercise, Exe.MSc: Patient-Exercise-Cell, Exe.MSc.Glu: Glucosamine-Exercise-Patient.a: Significance relative to healthy control, b: Significance relative to patient, c: Mean Glucamamine, d: significant relative to cell group, e: significant relative to exercise group.
Fig. 2. Comparison of mean CPII levels in different research groups.
Normal: Control-healthy, Patient: Control-patient, saline: Saline-patient, Exe: Exercise-patient, MSc: Cell-patient, Glu: Glucosamine-patient, MSc.Glu: Cell-glucosamine-patient, Glu.Exe: Patient-Glucosamine-Exercise, Exe.MSc: Patient-Exercise-Cell, Exe.MSc.Glu: Glucosamine-Exercise-Patient.a: Significance relative to healthy control, b: Significance relative to patient, c: Mean You have relative to glucosamine, d: significant relative to cell group.
Conclusion: The results of the present study showed that the expression of BSPII gene and CPII levels of cartilage tissue in the model of osteoarthritis mice increased abnormally. BSP is a bone-specific protein produced by osteoblasts. BSP is specifically enriched between the cartilage-bone phases (3). Propeptide C of type II collagen (CPII) also acts as procollagen by the activity of a proteinase C and is released to form fibrils. The peptide is released from the cartilage and enters the bloodstream. Therefore, its presence in body fluids is one of the indicators of collagen synthesis in cartilage (3). In the present study, CPII levels and BSPII gene expression were assessed at 8 weeks using a osteoarthritis model and the protective effect of exercise, glucosamine and stem cells. The results of our study showed that glucosamine consumption, stem cell injection, exercise or a combination of them effectively control cartilage depletion in a model of osteoarthritis mice. CPII levels and BSPII gene expression were reduced to normal by combined exercise + cell + glucosamine intervention. These values had the most significant decrease in the combined group compared to the osteoarthritis group. These results suggest that the use of exercise in combination with glucosamine intake and cell injection has a synergistic effect on cartilage protection. In the mouse osteoarthritis model, glucosamine inhibits type II collagen degradation by preserving proteoglycans and enhances type II collagen synthesis in articular cartilage. In addition, glucosamine in the early stages of experimental osteoarthritis has a positive effect on the regeneration, structure and mineralization of bone under cartilage and can increase the production of hyaluronic acid in synovial tissue (16). Overall, the findings showed that osteoarthritis increased the expression of BSPII gene and CPII levels in cartilage tissue and a combination of cell + glucosamine + exercise was more effective than exercise, cell and glucosamine alone to reduce these markers.
Type of Study: Research |
Subject:
Exercise Physiology
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