Research Article - Biomedical Research (2021) Volume 32, Issue 3
Beneficial effect of fasudil in osteoarthritis by enhancing the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells
- Corresponding Author:
- Qingyun Xue
Department of Orthopedics
Beijing Hospital National Center of Gerontology
Institute of Geriatric Medicine
Chinese Academy of Medical Sciences
Accepted date: June 10, 2021
Present report evaluates the effect of fasudil on the chondrogenic differentiation of BMSCs and on management of OA (in vivo and in vitro). Primary BMSCs were isolated from each rat for in vitro study and BMSCs were treated in presence and absence of fasudil. Cell viability assay was assessed by CCK-8 assay and chondrogenic differentiation and migration of BMSCs by incubating with fasudil for 14 days. In vivo OA was induced in rats by surgical procedure and treated with fasudil 30 mg/kg, p.o. Level of cytokines and expression of JNK, ERK and p38 protein were assessed in the cartilage tissue of OA rats. There was significant increase in the cell viability and migration in fasudil 100 nM treated BMSCs than fasudil 0 nM. Protein and mRNA expression of Sox9, Col2a1, Aggrecan and Col10a1 were significantly enhanced in fasudil treated 10 and 100 nM compared to 0 nM on day 7 and 14. Expression of JNK, p38 and ERK protein was reduced significantly (P<0.01) in the fasudil treated group than control group of BMSCs. The fasudil reduced cytokine level and TGF-β, and increased the expression of MMP-13, Col II in the cartilage tissue of OA rats. Moreover, treatment with fasudil ameliorates the histopathological changes in the cartilage tissue of OA rats. In conclusion, fasudil treatment protects the osteoarthritis by promoting the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Moreover, fasudil reduces the level of cytokine and activation of JNK/MAPK/ERK signaling pathways in the cartilage tissue of OA rats.
Osteoarthritis (OA) is a chronic joint disease which leads to hyperostosis and degradation of the articular cartilage . There are several etiological factors such abnormal loading, repetitive injury, aging, and obesity involved in the development of osteoarthritis . OA is one of the major causes of the disability throughout the globe, especially due to limitation of locomotor activity and pain in elderly patient. Differentiation and condensation of MSC into chondrocyte has role in the skeletal development of OA, commonly known as chondrogenesis . Adipocytes, osteoblasts, and chondrocytes like multi-differentiation ability of bone marrow MSCs in the field of orthopaedics therapy and research clinically . In OA, cartilage injury repairment stimulated by regulating the differentiation to chondrocytes from MSC and there are only few pathways involved in the regulation of it. Literature suggested several biological developments involve the regulation of MAPK/ERK pathway including activation of chondrogenic differentiation . Moreover, Transforming Growth Factor-β1 (TGF-β1) stimulates the proliferation and differentiation of chondrocytes by activating the MAPK/ERK pathway . Thus, molecules activating MAPK/ERK pathway need to explore on chondrogenic differentiation MSC for the treatment of OA.
Rho kinase (ROCK) inhibitor is clinically used for the treatment of subarachnoid haemorrhage  and Fasudil (FAS) is a ROCK inhibitor, which shows very few side effects . FAS normalize the blood flow and protect the damaged nerve tissues and thereby prevents spinal cord injury . Moreover, a study suggests that FAS enhances the differentiation of MSC by regulating the MAPK pathway for the treatment of spinal cord injury . Thus present report evaluates the effect of Fasudil on chondrogenic differentiation of BMSC and management of OA.
Materials and Methods
Sprague-Dawley rats (200-250 g) were housed and maintained as per international guidelines; the animal study was approved by institutional animal ethical committee of Beijing Hospital, China (IAEC/BH-NCG/03/2019).
Chondrocyte isolation and culture
Primary BMSCs were isolated as described previously from the cavities of the tibial plateaus and femora and cultured for 2 days at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with transforming growth factor-β-3 (TGF-β), sodium pyruvate, ascorbate, and dexamethasone. Non-adherent cells were removed during medium changes, which were performed until the cells attained 80-90% confluence. Flow cytometry was used to characterise the BMSCs in terms of typical surface markers. Antibodies against CD29, CD90, CD45, or CD105 were added to BMSCs, following the manufacturer’s directions.
Cell Counting Kit-8 (CCK-8) assay
CCK-8 assay was used as per the direction given by the manufacturer of the kit using cell viability assay. Cells (5 × 103 cells/well) were added in the 12 well plate and cells were treated with fasudil (0, 10 and 100 nM). Later each well plate is treated with CCK-8 solution (10 μL) and incubates at 37 °C for 1h with 5% CO2. Microplate Reader was used to determine the absorbance at a wavelength of 450 nm.
Transwell filter system was used to examine the cell migration. BMSCs (200 μL) suspended in serum-free medium used to fill the upper chamber and complete medium (600 μL) was used to fill the lower chamber. Humidity incubator was used to keep BMSCs containing Transwell filter system. Methanol was used to fix the BMSCs after 12 h of incubation at 37 °C and upper surface of the filter on which non traversed cells are present were removed using cotton swab. Crystal violet was used for 15 min to stain the lower side of the filter migrated cells microscope was used to estimate the count of migrated cells.
Chondrogenic induction of BMSCs
Induction of chondrogenic differentiation of BMSCs as per reported method. BMSCs (1 × 105 cells/well) were seeded into 12 well plate and cells were incubated till confluence reached 80% at 37 °C. chondrogenic-inductive medium was replaced in place of culture medium in presence and absence of fasudil. BMSCs kept for chondrogenic differentiation for 14 days and later collected BMSCs were placed for further study.
The Trizol reagent was used to extract total RNAs, followed by reverse transcription into cDNAs. mRNA expression of Sox9, Col2a1, Col10a1 and β-actin in BMSCs were derived with the aid of the SYBR Green gene expression assay. mRNA expressions were also analysed using a TaqMan microRNA assay kit.
Western blot analysis
RIPA lysis buffer was used to extract the protein from BMSCs and bicinchoninic acid Protein Assay Kit was used to determine the protein content. the proteins separated via 10% (w/v) Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes, which were blocked with 5% (w/v) blocking solution (non-fat milk) and incubated in blocking buffer with primary antibodies overnight at 4 °C. Goat secondary antibodies conjugated with horseradish peroxidase were added, and a chemiluminescence kit was used to detect the proteins.
In-vivo model of osteoarthritis
The animals were divided into normal, OA, and fasudil treated groups. All were injected with 7 mg/kg xylazine and 60 mg/kg of ketamine. To induce OA, the medial side of the patellar tendon was incised, the patella dislocated, and the anterior cruciate ligament transected with a surgical blade. The medial retinaculum was repaired, and the skin closed. The fasudil was orally at a dose of 30 mg/ kg. qRT-PCR was performed (as mentioned above).
Estimation of protein and cytokine levels
All animals were sacrificed via decapitation and articular cartilage isolated from the medial tibial plateaus. qRTPCR was used to quantify the levels of mRNAs encoding MAPK, p-ERK, ERK, p-JNK, JNK, MMP-3, and Col II in tissue homogenates. IL-1β, IL-6, and TGF-β levels were measured by ELISA.
Isolated knee joints were fixed in 10% (v/v) formalin for 3 days, placed into molten paraffin, and cut into 6 μmthick sections when the paraffin set. The sections were stained with Safranin-O and cartilage degeneration scored as described previously.
All data are expressed as means ± standard error mean (SEM; n = 10) and the statistical analysis consisted of a one-way Analysis Of Variance (ANOVA). Post-hoc comparisons of means were carried out with Dunnett’s post hoc test using GraphPad Prism software (ver. 6.1; San Diego, CA, USA). P values<0.05 were considered to indicate statistical significance.
BMSCs were assessed via flow cytometry (Figure 1 A-D). The cells were CD29-, CD90-, and CD105-positive and CD45-negative, indicating that they were BMSCs.
Fasudil enhances the migration and proliferation of BMSCs
Migration and proliferation of BMSCs was assessed in the fasudil treated cells as shown in Figure 2A and 2B. CCK- 8 assay was used to determine the viability of BMSCs in fasudil treated cells on day 1, 3 and 7. There was no significant change observed on the viability of fasudil treated cells on day 1 and 3. However viability of cell was enhanced in fasudil treated 100 nM compared to 0 nM cells (Figure 2A). Transwell assay was used to determine the migration of BMSCs in fasudil treated cells as shown in Figure 2B. There was significant (P<0.01) increase in the migration of cells in fasudil treated 100 nM compared to 0 nM cells.
Fasudil enhances the chondrogenic differentiation of BMSCs
Effect of fasudil on the chondrogenic differentiation of BMSCs was assessed by determining the protein and mRNA expression of Sox9, Col2a1, Aggrecan and Col10a1 in the BMSCs on day 3, 7 and 14 (Figure 3A-D). Protein and mRNA expression of Sox9, Col2a1, Aggrecan and Col10a1 were not altered on day 3 of fasudil treated BMSCs. However, the protein and mRNA expression of Sox9, Col2a1, Aggrecan and Col10a1 were significantly enhanced in fasudil treated 10 and 100 nM compared to 0 nM on day 7 and 14.
Figure 3. Effect of fasudil on the chondrogenic differentiation of BMSCs. A: Protein and mRNA expression of Sox9; B: Protein and mRNA expression of Col2a1; C: Protein and mRNA expression of Aggrecan; D: Protein and mRNA expression of Col10a1.
Mean ± SEM (n=06); #P<0.05, ##P<0.01, ###P<0.001 compare with 0 nM concentration of fasudil.
Fasudil ameliorates the JNK/MAPK/ERK pathway
Expression of JNK, p38 and ERK proteins were estimated in the fasudil treated BMSCs by using western blot assay as shown in Figure 4. There was significant increase (P<0.01) in the expression of JNK, p38 and ERK protein in the negative control group compared to control group of BMSCs. Expression of JNK, p38 and ERK protein was reduced significantly (P<0.01) in the fasudil treated group than control group of BMSCs.
Fasudil reduces the level of inflammatory cytokines
Effect of fasudil was estimated on the level of inflammatory cytokines in the cartilage tissue of OA rats by ELISA as shown in Figure 5. Level of IL-1β, IL-6 and TGF-β1 was enhanced in the cartilage tissue of OA group than normal group of rats. There was significantly (P<0.01) reduction in the level of IL-1β, IL-6 and TGF-β1 in the cartilage tissue of fasudil treated group than OA group.
Fasudil ameliorates the expression of JNK, ERK, p38, MMP-13 and Col II
mRNA expression of JNK, ERK, p38, MMP-13 and Col II was observed in the cartilage tissue of fasudil treated OA rats (Figure 6). There was significant increase in the mRNA expression of JNK, ERK and p38 and reduction in the mRNA expression of MMP-13 and Col II in the cartilage tissue of OA group than normal group of rats. Treatment with fasudil ameliorates the mRNA expression of JNK, ERK, p38, MMP-13 and Col II in the cartilage tissue of OA rats.
Fasudil ameliorates the histopathological changes in the cartilage tissue of OA
Histopathological changes were assessed in the cartilage tissue of fasudil treated osteoarthritis rat by using H&E staining as shown in Figure 7. H&E staining suggest shows the sever erosion in the cartilage tissue of OA group than normal group of rats. However, treatment with fasudil attenuates the erosion to the cartilage tissue of OA rats.
Osteoarthritis is a major form of arthritis and chronic used drugs for the management of it has several limitations. Excessive joint loading, foreign body intrusion and inflammation are the major causes of damage to chondrocytes in OA . Cellular density reported to be reduced, which inhibits the proliferation of chondrocytes and chondrocytes are not having ability to regenerate by themselves . Although many of the treatment concentrate on cartilage engineering but insufficient regeneration can achieve. BMSCs differentiate into myocytes, osteoblasts, and chondrocytes  and thus present report evaluates the effect of fasudil on the chondrogenic differentiation of BMSCs and on management of OA (in vivo and in vitro).
Sox family is a transcription factor involved in the cellular development and differentiation . There are several growth and transcription factors regulate the chondrogenic differentiation of BMSCs. Sox9 is reported to be a marker of osteogenesis and chondrogenesis of BMSCs by regulating the differentiation and condensation of it . Chondrogenesis occur due to increase in the expression of Sox9 by activating the Col2a1 transcription and activation of expression of collagen (Col10a1) gene, which enhances the chondrogenesis . Moreover, chondrocyte synthesizes proteoglycans by regulating the Aggrecan. Literature suggest that chondrogenic differentiation activates by enhancing the expression of Sox9  and data of the study also suggest that treatment with fasudil modulates the expression of Sox9, Col2a1, Col10a1 and Aggrecan.
Moreover, proliferation and migration of BMSCs enhances in fasudil treated group than control group. Cartilage destruction in OA is attributable to increased cytokine levels . The cytokines reduce the expression levels of MMP-13, Col II, and TGF-β, all of which stimulate chondrocyte differentiation . The fasudil reduced cytokine level and TGF-β, and increased the expression of MMP-13, Col II in the cartilage tissue of OA rats.
MAPK containing JNK, p38 and ERK pathway involved in the normal biological processes including activation of mitochondrial pathways . Moreover, number of types of tumors induces the autophagic cell death by JNK pathway. JNK/MAPK/ERK signalling pathways activation involved in the chondrogenic differentiation . Treatment with fasudil ameliorates the altered JNK/ MAPK/ERK signaling pathways in BMSCs and in the cartilage tissue of OA rats.
In conclusion, fasudil treatment protects the osteoarthritis by promoting the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Moreover, fasudil reduces the level of cytokine and activation of JNK/MAPK/ERK signaling pathways in the cartilage tissue of OA rats.
All the authors of presented manuscript thankful to Fundamental Research Funds for the Central Universities (No: 3332018176) for providing financial support for the conduct of this work.
Conflict of Interest
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