| Neck and low back pain is the most common musculoskeletal complaint, and statistics showed that 80% of the population will experience it over the course of their lifetime. Intervertebral disc degeneration (IVDD) is an important factor in the pathogenesis of neck and low back pain, but the mechanism of degeneration is still unclear yet. Studies on the mechanism of IVDD and methods of inhibiting it become a research hotspot.Objective:The lack of blood vessels and scarcity of cells make intervertebral disc can hardly repair itself.Bone marrow derived mesenchymal stem cells (BMSCs) can promote the regeneration of intervertebral disc tissues, which has been reported by massive experimental studies and some clinical studies. Due to the limited source of BMSCs as well as operative trauma of implantation, a lot of studies focuse on promoting the homing of endogenous BMSCs. The effectiveness of "Bushen Huoxue Shujin Recipe" in treating degenerated disc disease(DDD) has been proved by previous experimental researches and clinical trials,which demonstrated that it can inhibit inflammation reaction through regulating Nuclear factor-κB(NF-κB)to delay IVDD. Therefore, we proposed a hypothesis that "Bushen Huoxue Shujin Recipe" can promote the homing of BMSCs to the degenerated disc through regulating NF-κB/CXCR4 signal.Based on this hypothesis, we carried out the following experimental study to observe the effect of "Bushen Huoxue Shujin Recipe" on the migration of BMSCs as well as the expression of NF-κB and CXCR4 in vitro experiment and explore the molecure mechanism.Methods:The femurs were removed from four-week-old SD rats of SPF level and BMSCs were isolated by whole bone marrow adherence method,then plated onto T25 cm2 flasks and cultured with α-MEM medium with GlutaMAX-I supplemented with 10% fetal bovine serums while observed underinverted phase contrast microscope. The 3rd generation of rBMSCs (P3) were tested for their ability to differentiate into adipogenic and osteogenic and comfirmed with oil red O and alizarin red S staining.For preparation of serum containg high, middle and low concentrations of "Bushen Huoxue Shujin Recipe",20 SD adult rats were randomly divided into four groups:the low-dosagee、middle-dosage and high-dosage groups were treated with prepared "Bushen Huoxue Shujin" Decoction for two weeks, and the control group was treatd the double distilled water.HPLC and MS were adopted to analyze the fingerprints of TCMs and drug serum in quality control.Then, we observed the proliferation of P3 BMSCs cultured and induced by drug serum and blank serumfor 24h by Cell Counting Kit-8 (CCK-8)assay,and observed the migration of P3 BMSCs cultured and induced by drug serum and blank serum for 48h by Transwell Cell Migration Assay,as well as observed the effect of drug serum and blank serum on the expression of NF-κB and CXCR-4 proteins by Western bloting.Results:1. Morphological observation and identification of rBMSCs:After cultured for 5-7 days,therBMSCs obtained passage and proliferatied faster than before.It took 3 days that passage cells coat 80% of the whole bottom of bottle and fused. The 3rd、4rd and 5rd generation cells were of high purities and proliferation.BMSCs were spindle-shaped or polygons-shaped, spiral shaped arrangement with more cells. For adipogenic differentiation, the adipogenic induction media was changed every 3 days until fat droplets appeard.Cell differentiation to adipocyte was confirmed by Oil Red O staining and visualized by light microscopy.For osteogenic differentiation, the differntiation medium were changed every 3 days.Calcium deposition in the extracellular matrix was determined by Alizarin Red S staining after 14 days of osteogenic differentiation.2. Preparation of serum containing "Bushen Huoxue Shujin Recipe":Twelve kinds of Chinese Herbal Monomerwere(CHM) were detected in TCM decoctionand 7 of which were detected in serum by LC-MS. Ferulic acid (Ligusticum wallichii, Angelica sinensis), Asperosaponin (Radix dipsaci) and protocatechuic acid (Rhizoma cibotii) had the highest concentration in drug serum than others,and the concentration of them decreased from high, middle to low concentration of drug serum.3. Effect of "Bushen Huoxue Shujin Recipe" on the proliferation and migration of BMSCs: After 24h, the result of Cell Counting Kit-8 (CCK-8)assay showed that there was significant difference between drug serrm groups and control groupon cell survival rate (P<0.05). But there was no significant difference between drug serm groups (P>0.05).The Transwell Cell Migration Assay showed that, cells could be observed in the downside of upper chamber in each group, which suggested that the isolated rBMSCs had migration behavior. Compared with control group, the number of migration cells in low concentration of TCM group was larger, but the difference was not statistically significant (P>0.05). The number of migration cells in middle and high concentration of drug serum groups were significantly larger than that of control group while high concentration group showed the largest number, and the difference was statistically significant (P<0.05),by the same time there was significant difference between drug serum groups (P<0.05).4. Effect of "Bushen Huoxue Shujin Recipe" on the expression of NF-κB and CXCR-4 proteins:The result of Western bloting showed that NF-κB and CXCR-4 proteins were expressed in each experimental group, but compared with control group, drug serum groups had higher expression levels of NF-κB and CXCR-4 proteins, and the difference was statistically significant (P<0.05).In addition,the difference between drug serum groups was statistically significant(P<0.05).Conclusion:"Bushen Huoxue Shujin Recipe" may induce the migration of BMSCs through regulating the expression of NF-κB and CXCR-4. |
PDF Full Text Request