| Background:Epidemiological survey results showed an increasing incidence of low back pain with the upgrading of people’s life pace and work intensity. The lumbar is the most force and move movement of body, which provides supporting for the human body and muscles,and protection of abdominal viscera, and more easy to be injured. In the outpatient about60%of the patients are suffering from vary degrees of low back pain. The causes of lumbar pain are about more than ten, and the most common reasons include degenerative ones:such as lumbar muscle strain, degenerative ones:lumbar disc herniation (LDH), stenosis of the spinal canal and hyperosteogeny, tumor ones:lumbar tumors and tumors from vertebral canal, immunity ones:ankylosing spondylitis, traumatic ones:lumbar fracture and soft tissue injury, disco-genic low back pain (DLBP) is the hot spot of modern orthopedic research with its complex etiology, unclear pathogenesis and absence of effective clinical treatment.Electrophysiological studies indicated that, under normal physiological conditions, nerve fiber only exists at the outer layer of the fibrous ring of intervertebral disc, while constitutively distributed in the nucleus pulposus and the annulus inner layer in DLBP patients. This evidence suggests that the ingrowth of nerve fibers is one of the important pathogenesis mechanisms associated with DLBP. Nerve growth factor(NGF) overexpression and excessive secretion of inflammatory factors such as interleukin-1β(IL-1β) and interleukin-6(IL-6) is the key in the DLBP pathogenesis. Many people believed that NGF abnormal expression is considered the major pathological mechanism of nerve in-growth in DLBP, and inflammatory pain signaling in DLBP is mainly mediated by NGF-dependent neurons. NGF can not only induce the nerve in-growth in DLBP, but also involves inflammatory pain sensitization.Therefore, we hypothesized that NGF abnormal expression caused by IL-1β and IL-6is an important mechanism of intervertebral disc injury. Based on the above speculation, we preliminarily determined the experiment to observe whether small interfering RNA (siRNA) has interference effects on the NGF in the presence of inflammatory cytokines IL-1β and IL-6.Objective:To investigate the interference effect of siRNA on NGF induced by inflammatory factor IL-1β and IL-6so as to provide novel targets for clinical treatment of DLBP.Methods:Sixty Sprague-Dawley (SD) rats, of specific pathogen free level; half male and half female; weighing180-240g, mean (223±12.4) g; aged2.5-3.5months, mean (2.94±0.73) months; were provided by the Animal Center of Shandong University School of Medicine, China with license No. SYXK (Lu)2007-0081.Isolation of nucleus pulpous and annulus fibrosus cells from intervertebral disc of SD rats. The intervertebral disc was rinsed with ice-cold PBS three times immediately after removal, the nucleus pulposus tissue was isolated and cut into pieces in DMEM/F12medium, then the pieces were centrifuged at4000r/min,4℃and resuspended, incubated at37℃with5%CO2for8hours. The supernatant was removed after centrifugation at4000r/min,4℃, the cells were rinsed with PBS and digested with0.25%trypsin and2%Ⅱ collagenase. After another4℃centrifugation at4000r/min, the supernatant was removed and the cells was resuspended in DMEM/F12medium, then the resuspended cells were counted to determine the number of cells up to104/L. Subsequently the cells at1x104/L were transferred to5%CO2incubator and cultured37℃. After cells were cultured for7days, the cells were transferred to serum-free medium for additional24hours. The cells were divided into a control group and an experimental group, culturing in DMEM/F12medium containing0.3%fetal bovine serum, experimental group was added with10nmol/L,20nmol/L,50nmol/L,100nmol/L of interleukin-6and interleukin-1β for48hours. Cells at the logarithmic growth phase were incubated onto the six-well culture plate at the density of2.5x105/mL. In accordance with the kit instructions, DEPC H2O and Lipofectamine TM2000were diluted and then mixed with NGF-siRNA, the mixture was incubated for10minutes and added to the culture plate (2.5×105/hole) in37℃,5%CO2incubator. The culture medium was changed promptly. The conversion rate of the cells was determined with flow cytometry to assess transfer efficiency. Reverse transcription and RT-PCR amplification were automatically performed with by PCR instrument, and paired using a conventional system. Reaction conditions:Reverse transcription:7℃for15min,85℃for5s; amplification:93℃for2min;93℃for15s,55℃for25s,72℃for25s, totally40cycles;72℃for10min. Ct value (the number of copies/μl cDNA) refers to the number of cycles when fluorescence signal reaches the preset threshold value, Ct values for each template has a log-linear relationship with the initial copy number, i.e., the higher the initial copy number is, the smaller Ct value is. Due to the difference of total RNA concentration in different samples, NGF mRNA expression is calculated according to the following formula:Cttarget gene/Ctreference gene.Cell culture medium100μL were collected to measure NGF levels in strict accordance with the ELISA kit instructions. Before and after which the NGF mRNA expression was detected by real-time Q-PCR and the NGF content was detected by ELISA. Data were analyzed using SPSS17.0software. NGF mRNA expression and NGF levels were normally distributed and expressed as mean±SD. The difference before and after the introduction NGF-siRNA was compared with paired t-test. Multiple-group comparison was performed using one-way analysis of variance, a=0.05was considered as the standard.Results:Cells were successfully cultured in vitro, nucleus pulposus cells and fibrous annulus cells were obtained. The standard amplification curve at different concentrations showed good parallelism and surface smooth, indicating that the amplification conditions were accurate with a high specificity. The linear regression graphs have a good regression and high reproducibility, and the regression coefficient R2was>0.99.It was showed by the flow cytometry instrument test results that the NGF-siRNA cell conversion rate was99.8%. Before the NGF-siRNA intervention, NGF mRNA expression in10nmol/L,20nmol/L,50nmol/L,100nmol/L groups was increased3.4,3.7,4.7,8.0times compared with the control group. After NGF-siRNA intervention, NGF-mRNA relative expression in each group was decreased39.5%,45.5%,45.3%,47.8%compared with before intervention (P<0.05). Before NGF-siRNA intervention, NGF levels in10nmol/L,20nmol/L,50nmol/L,100nmol/L groups were increased2.9,3.3,4.5,7.4times compared with the control group; after NGF-siRNA intervention, NGF levels in each group were decreased33.8%,35.4%,43.0%,54.9%compared with before intervention (P<0.05).Conclusion:This finding indicates that, siRNA can inhibit the stimulation effect of pro-inflammatory cytokines IL-1βand IL-6on NGF in intervertebral disc cells, which provide a novel approach for the treatment of DLBP. IL-1βand IL-6could stimulate NGF on intervertebral disc cells in vitro culture model and its efficiency has concentration dependence.Contribution:From the angle of the molecular biology, we can lead the NGF-siRNA into the discs of DLBP patients, which can inhibit the effect of IL-6and IL-1β, then inhibit the nerve fibers ingrowth, and cut the transmission to the brains. A new approach to the treatment of DLBP is our research most contribution.Innovation:Although we used the traditional way to isolate and culture cells, but the most different things from the other experimental groups was that there were4concentration groups, by which we could observe the change of the corresponding index how to happen under different conditions better to find the rules. Although more difficult to experiment, but closer to the law of development of disease ranging from mild to severe.Inadequate:However, the limitations of this study lies on the use of cultured rat intervertebral disc cells in vitro. But the effect in the human body needs further study to investigate, while the siRNA clinical feasibility and ethical things still need to determine.The exact mechanism associated with DLBP is very complex, and most of affected patients suffer from a long-term, chronic lesion. Therefore Freund’s adjuvant-caused inodel of inflammatory disc only partially mimics inflammatory reaction in the DLBP, and the establishment of models needs further improvement. On the other hand, the present study only confirmed the in vitro applicability of NGF-siRNA interference in the treatment of DLBP, and further investigations are needed for in vivo tests. |
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