Reactive oxygen species (ROS) are thought to have a strong correlation with a number of intervertebral disc (IVD) diseases. Here, we aimed to determine whether ROS represent an etiology of low back pain (LBP) during IVD degeneration. Thirty degenerated intervertebral disc samples were obtained from patients, and ROS levels were quantified using dihydroethidium (DHE) staining. The results suggested a significant correlation between the ROS level and the severity of LBP. Subsequently, a puncture-induced LBP model was established in rats, and ROS levels significantly increased compared with those in the sham surgery group, accompanied with severe puncture-induced IVD degeneration. In addition, when ROS levels were increased by H2O2 administration or decreased by NAC treatment, the rats showed increased or decreased LBP, respectively. Based on this evidence, we further determined that stimulation with H2O2 in nucleus pulposus cells (NPCs)
Low back pain (LBP) is a serious chronic disease that reduces quality of life and increases psychological burden in patients. Accordingly, approximately one-quarter of U.S. adults reported having LBP lasting at least 1 whole day in the past 3 months, and 7.6% reported at least 1 episode of severe acute low back pain within a 1-year period [
There is no doubt that the intervertebral discs (IVDs) play a critical role in maintaining the stability of the whole spine, and abnormal anatomy or physiological dysfunction of IVDs leads to a series of spine-related diseases, especially LBP [
Reactive oxygen species (ROS) are products of biomarkers expressed in response to cellular damage or stimuli. Nucleus pulposus cells (NPCs) are the core of the IVDs, and they exhibit a remarkable increase in ROS levels after exposure to mechanical stress [
Until now, there has been no evidence concerning the pathological role of ROS in LBP. Thus, the first aim of this study was to elucidate the potential relationship between ROS and LBP. In addition, we wanted to investigate whether substance P (SP), a peptide in the IVDs that is responsible for LBP, participated in ROS-mediated LBP. The elimination of ROS in the IVDs could provide a promising therapeutic method for LBP in the future.
The study was authorized by the Institutional Review Board of Ruijin Hospital, Shanghai Jiaotong University School of Medicine, and every participant signed an informed consent form. Thirty patients who underwent posterior lumbar discectomy at our hospital because of lumbar intervertebral disc degeneration were enrolled in this study between May 2020 and September 2020. All patients had LBP accompanied with or without sciatica for at least 6 months and failed to conservative treatment or physical therapy. If the patients had lumbar disc herniation associated with Modic changes, spondylolisthesis, or spinal instability, instrumented posterior lumbar interbody fusion was performed at the same time. The included patients ranged from 36 to 82 years of age, with an average age of
All animal experiments in this study were approved by the Animal Care and Use Committee of Henan Provincial Orthopedic Institute, and we followed the protocols of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Based on a previous study [
LBP was quantified via the mechanical and/or cold paw/foot withdrawal threshold method following previous reports [
To assess cold allodynia, 100% acetone was used [
After extraction, total proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (0.45
The Trizol reagent (Invitrogen, Life Technologies Corporation, CA, U.S.) was used to extract total RNA, and cDNA was synthesized from 1
To quantify the ROS levels in the IVDs of patients, the samples were frozen at -20°C and sectioned at 5
For IHC analysis, the human nucleus pulposus tissue was fixed with 4% paraformaldehyde for 12 h and then processed via routine paraffin embedding, sectioning, and deparaffinization. The IVDs from rats were fixed with 4% paraformaldehyde for 12 h and then were decalcified using 10% ethylenediaminetetraacetic acid (EDTA) for 1 month before sectioning. Subsequently, the sections were incubated with a TAC1/substance P rabbit polyclonal antibody (cat. no. AF8094, Beyotime Biotechnology Inc., Shanghai, China) at 4°C overnight. A specific IHC kit (cat. No. K5007, Agilent DAKO Inc., CA, US) was used for the whole process according the manufacturer’s protocol. Nuclei were counterstained with hemalum (cat. G1004, Servicebio Inc., China). The stained samples were observed and photographed under a microscope (Axio, Carl Zeiss, Oberkochen, Germany).
The data are expressed as the
To investigate the correlation between LBP and ROS, we performed DHE staining of IVDs from patients and quantified the ROS levels by measuring the mean density (Figure
The severity of LBP showed a correlation with ROS levels in human IVDs. (a) DHE staining suggested a gradual increase in ROS levels along with more severe LBP in patients. (b, c) Linear regression analysis suggested a significant positive correlation between the ROS level in the NP and the VAS score or ODI score. (d) There was a significant negative correlation between the ROS level and the JOA score. (e) When the patients were classified as having mild, moderate, and severe LBP, the ROS levels were significantly gradually increased (
To verify the pathological role of ROS in LBP, we established an LBP model in rats according to a previous report [
Increased ROS levels determined the severity of LBP in rats. (a) Immunofluorescence analysis of DHE suggested an increase in ROS after puncture of IVD with an 18-gauge needle. (b, c) Administration of H2O2 after puncture of IVDs resulted in much more severe LBP, indicating a decrease in the mechanical allodynia threshold and an increase in the cold allodynia threshold when compared with those of the puncture + saline group. (d, e) Administration of NAC (a classical antioxidant) significantly alleviated LBP in rats, suggesting an increase in the mechanical allodynia threshold and a decrease in the cold allodynia threshold compared with those of the puncture + saline group. (f) The timeline of drug delivery and behavioral testing for all groups (The data are shown as the
It was suggested that SP, a peptide thought to be involved in the synaptic transmission of pain, could be secreted by NPC and play a critical role in discogenic LBP. Here, the relative gene expression of SP significantly increased when NPCs were cocultured with H2O2 in a dose-dependent manner, as shown in Figure
ROS significantly induced the expression of substance P. (a) H2O2 significantly induced the expression of the SP gene in NPCs in a dose-dependent manner with RT-PCR expression. (b) Western blot analysis also verified the increase in SP after stimulation with H2O2 in a dose-dependent manner. (c) Administration of H2O2 into the IVDs of rats resulted in significant upregulation of the SP protein. (d) IHC analysis suggested a remarkable increase in SP protein levels in the NP after administration of 100
To further confirm the pathological role of SP in LBP, we quantified the expression of SP in human IVDs. The expression of SP gradually increased with increasing severity of LBP, with a statistically significant dose-dependent response (as depicted in Figure
SP was the key factor mediating LBP. (a) IHC analysis suggested an increase of SP levels in human NP tissue, with a significant difference among the VAS mild, VAS moderate, and VAS severe groups. (b, c) Administration of SP into the IVDs of rats significantly induced LBP, indicating a decrease in the mechanical allodynia threshold and an increase in the cold allodynia threshold when compared with those of the puncture + saline group, with a dose-dependent response. (d, e) By contrast, administration of the SP receptor inhibitor of aprepitant significantly attenuated LBP in rats. (f) The timeline of drug delivery and behavioral testing for all groups (The data are shown as the
Here, we demonstrated that the ROS level in IVDs had a correlation with LBP based on clinical and animal studies. In addition, increased ROS levels resulted in significant upregulation of SP, which is a crucial factor in inducing LBP in IVDs. In contrast, elimination of ROS or inhibition of the SP receptor induced remarkable relief of LBP in patients. Overall, we drew the reasonable conclusion that increased ROS levels acted as the trigger for LBP by upregulating SP in IVDs.
Multiple factors lead to an increase in ROS levels in IVDs. A previous study suggested that
Although there are few reports clarifying the relationship between ROS and LBP, ROS are thought to be a key factor in inducing neuropathic pain or inflammatory pain. For example, control of ROS levels attenuated neuroexcitability and restrict bidirectional signaling between neurons, glia, and immune cells that creates and amplifies pain [
There was no previous report clarifying how ROS induce LBP. Here, our data suggested that SP maybe a key factor participating in ROS-induced LBP. Many studies have suggested that SP is an independent risk factor for LBP, because SP is a critical neurotransmitter peptide that promotes pain transmission in nerves. Previous histological finding suggested that nerve growth into IVD with expression of SP was a key factor in the pathogenesis of chronic low back pain [
Here, we not only proved the close relationship between SP and LBP but also identified NPCs as the main source of SP after ROS stimulation. It has been reported that NPCs secrete SP when stimulated by
However, there were still some limitations in this study. Firstly, the long-term therapeutic effectiveness of NAC or aprepitant treatment was not investigated in this study, and whether they were effective for chronic LBP was still unclear. In addition, the small clinical sample is another limitation in this study and that may be the reason for the low
In conclusion, ROS is a primary factor in the induction of LBP based on clinical and animal data, and the mechanism involves ROS-mediated stimulation of NPCs to secrete SP, which is a critical neurotransmitter peptide, to promote pain transmission within IVDs. Therefore, reducing the level of ROS with specific drugs may be an alternative method to treat LBP in the clinic.
Reactive oxygen species
Low back pain
Intervertebral disc
Dihydroethidium
Nucleus pulposus cells
Substance P.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
Jiancheng Zheng and Jian Zhang contributed equally to this work.
This work was supported by grant from the fund of the Guangci Youth Plan of Ruijin Hospital (No. GCQN-2019-A09), the National Natural Science Fund of China (NSFC no. 81702188), the National Natural Science Fund of China (NSFC no. 82004397), and the Major Project of TCM Research in Henan Province (Nos. 2019ZYZD02 and 20-21ZYZD12). We also thank Pro Nicole R, from AJE, for editing the English text of a draft of this manuscript.
The IVD of L4-5 had remarkable degeneration after an 18-gauge needle penetration. (a) The lateral X-ray examination demonstrated that an 18-guage needle was penetrated into the IVD of L4-5 during the surgery (marked with red arrow). (b) Lateral X-ray examination figured out the punctured intervertebral disc height decreased when compared with sham-surgery animal after two weeks penetration (marked with red arrow). (c) The punctured IVD had decreased gray value at short T1 inversion-recovery (STIR) sequence of MRI after two weeks when compared with sham-surgery IVD (marked with red arrow). (d) Histological and immunohistochemical analysis suggested that the IVD in sham-surgery had normal appearance, indicating as aggrecan-rich, bulging nucleus pulposus with rare proliferated chondrocytes and no clefts, and organized annulus fibrosus as discrete fibrous lamellae, while the penetrated IVD had disappearance of notochordal cells and the numerous levels of proliferated chondrocytes in nucleus pulposus area and the disruption of endplates and annulus fibrosus due to the needle penetration.