Abstract
Objectives Aberrant transforming growth factor β (TGFβ) activation is detrimental to both nucleus pulposus (NP) cells and cartilage endplates (CEPs), which can lead to intervertebral disc degeneration (IDD). Ligustrazine (LIG) reduces the expression of inflammatory factors and TGFβ1 in hypertrophic CEP to prevent IDD. In this study, we investigate the effects of LIG on NP cells and the TGFβ signaling. Design LIG was injected to the lumbar spinal instability (LSI) mouse model. The effect of LIG was evaluated by intervertebral disc (IVD) score in the LSI mouse model. The expression of activated TGFβ was examined using immunostaining with pSmad2/3 antibody. The upright posture (UP) rat model was also treated and evaluated in the same manner to assess the effect of LIG. In ex vivo study, IVDs from four-week old mice were isolated and treated with 10−5, 10−6, and 10−7 M of LIG. We used western blot to detect activated TGFβ expression. TGFβ-treated human nucleus pulposus cells (HNPCs) were cotreated with optimized dose of LIG in vitro. Immunofluorescence staining was performed to determine pSmad2/3, connective tissue growth factor (CCN2), and aggrecan (ACAN) expression levels. Results IVD score and the percentage of pSmad2/3+ NP cells were low in LIG-treated LSI mice in comparison with LSI mice, but close to the levels in the Sham group. Similarly, LIG reduced the overexpression of TGFβ1 in NP cells. The inhibitory effect of LIG was dose dependent. A dose of 10−5 M LIG not only strongly attenuated Smad2/3 phosphorylation in TGFβ-treated IVD ex vivo but also suppressed pSmad2/3, CCN2, and ACAN expression in TGFβ-treated NP cells in vitro. Conclusions LIG prevents IDD via suppression of TGFβ overactivation in NP cells.
Highlights
Nucleus pulposus (NP) cells reside in the center of intervertebral disc (IVD) and play a key role in development of intervertebral disc degeneration (IDD) [1]
LIG Maintains NP Cell Vacuoles and IVD Score in lumbar spinal instability (LSI) Mice. e effect of LIG on NP cells in vivo was assessed by using the LSI mice model
Safranin O staining showed reduced vacuole sizes in NP cells after LSI surgery, whereas in the case where LSI mice were treated with LIG, the vacuole sizes were similar to those in Sham mice (Figure 1(c)). e maintenance of IVD score and NP cells’ vacuole sizes indicated LIG preventing IDD progression
Summary
Nucleus pulposus (NP) cells reside in the center of intervertebral disc (IVD) and play a key role in development of intervertebral disc degeneration (IDD) [1]. Mechanical loading triggers IDD by accelerating NP shrinkage and subsequently transits to fibroblast-like cells, which have decreased capacities in maintenance of IVD plasticity [2]. Two clinical trials (680 participants) found a compound that contains Chuanxiong, which relieved neck pain better in the short term than placebo in cervical IDD patients. One trail (60 participants) indicated an oral herbal formula with the component of Chuanxiong, which relieved pain better than Mobicox or Methycobal in treatment for cervical IDD [4]. An animal experiment revealed herb formula “Fufangqishe-Pill,” which prevents upright posture(UP-) induced lumbar IDD in the rat model [5]. Further study showed the main effective component of “Fufangqishe-Pill,” LIG, decelerates the progression of lumbar IDD through inhibiting inflammatory factors such as IL-1β, COX-2, and iNOS in the upright posture (UP) rat model [6]. Since IDD is considered as a transition of fibrosis, we hypothesize
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