Ligustrazine Prevents Intervertebral Disc Degeneration via Suppression of Aberrant TGFβ Activation in Nucleus Pulposus Cells

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.


Introduction
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].
Ligustrazine (LIG) is an extract from Ligusticum wallichii Franchat (Chuanxiong), which is a traditional Chinese herb known for its wide usage in treating spinal degenerative diseases [3]. 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]. In addition to anti-inflammation effect, a standard traditional Chinese medicine library screening showed LIG exerts an antifibrosis effect via inhibition of TGFβ [7]. Since IDD is also considered as a transition of fibrosis, we hypothesize that prevention of IDD by LIG is related to regulation of TGFβ signaling.
Our previous work found the overexpression of TGFβ accelerates cartilage endplate (CEP) hypertrophy and NP cell dysfunction, leading to IDD [2,8]. Interestingly, LIG prevents CEP outgrowth and suppresses TGFβ1 expression in hypertrophic CEP of IDD rats [9]. However, whether LIG has an antifibrotic effect on NP cells is not known. To investigate the effects of LIG on NP cells and explore its potential mechanism involving in TGFβ signaling, we employed two IDD animal models to investigate the effects of LIG on NP cells and TGFβ levels in different species and different mechanical loading patterns. We then isolated IVD from 4-week old mice and tested the dose response of LIG on TGFβ signaling by western blot ex vivo. e optimized dose of LIG was further applied to NP cells in vitro to determine the TGFβ signal downstream target gene expression of CCN2 and ACAN.

LIG Preparation.
Ligustrazine phosphate (purity > 99%, MW: 252.21) was purchased from the Chinese Medicine and Biological Products Institute (Beijing, China). e solutions of ligustrazine phosphate were prepared in dimethylsulfoxide (DMSO, Sigma, USA) and diluted with culture media for the in vitro experiments. e final concentration of DMSO was no more than 0.1%.

Animal Models and LIG Administration.
After intraperitoneal injection, anesthetized with ketamine (80 mg/ kg) and xylazine (5 mg/kg), the 3rd-5th lumbar (L3-L5) spinous processes along with the supraspinous and interspinous ligaments of the 8-week old male C57BL/6J mice (28 g ± 5 g, Shanghai Laboratory Animal Center, China) were resected to induce the lumbar spine instability (LSI) mouse model. Sham operations were carried out only by detaching the posterior paravertebral muscles from the L3-L5 vertebrae. e operated mice were then intraperitoneally injected with ligustrazine hydrochloride (Nanning Maple Leaf Pharmaceutical Co., Ltd, China, 1 mL, 2 mg/d/per mouse) while the mice operated in the sham surgery were injected with the equal volume of saline solution at the same frequency and duration (once a day for one month). Protocol #PZSHUTCM190315024 was approved by the Shanghai University of Traditional Chinese Medicine Animal Care and Use Committee.
Sprague-Dawley (SD) (male, 1-month old, 200 g ± 20 g) rats (Shanghai Laboratory Animal Center, China) were anesthetized with 100 mg/kg ketamine by intraperitoneal injection. e forelimb surgery and customized cages force them to stand up which triggers IDD initiation as previously described [10]. Ligustrazine hydrochloride was administrated as described above (16 mL/kg·d, 4 mg/mL). eir lumbar spines were later dissected for further analysis. Protocol #PZSHUTCM190315024 was approved by the Shanghai University of Traditional Chinese Medicine Animal Care and Use Committee.
All animals were randomly divided into each group. All surgeries were performed in the laboratory, and drug administration was done in the animal facility. Animals were sacrificed before sample collection with CO 2 exposure followed by cervical dislocation.

Quantitative Histomorphometric Analysis.
Quantitative histomorphometric analysis was conducted with Image-Pro Plus software version 6.0 (Media Cybernetics Inc., Rockville, MD, USA). IVD scores were obtained as described previously [11]. In detail, the organization of annulus fibrosus (AF), border between the AF and NP, cellularity of the NP, and matrix of the NP were assessed. e percentage of pSmad2/3-positive cells was obtained by counting the number of positive staining cells and the total number of cells in the NP region. e area of TGFβ1-positive staining was calculated by measuring the positive staining area of region of interest that covers all cells in NP. e area of CCN2+ or ACAN+ per cell was measured by dividing the total positive staining area of CCN2 or ACAN+ by total cell numbers. Dividing the total positive staining area by total cell nucleus area, we get the percentage of pSmad2/3+ area per cell.

Western Blot.
e cellular extraction from NP tissue in ex vivo assay was analyzed with a western blot. e cellular extraction was centrifuged, and the concentration of supernatants was evaluated by the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and then the proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted on a polyvinylidene fluoride membrane (Bio-Rad Laboratories). After incubation in antibodies, proteins were detected using an enhanced chemiluminescence kit (Amersham Biosciences, Pittsburgh, PA, USA). e target protein expression was examined with the following anti- Cell Signaling Technology Inc.), and GAPDH (1 : 1 000, 8884, Cell Signaling Technology Inc.).

Statistical
Analyses. e data are expressed as means ± standard error (SE), and statistical significance was calculated using one-way analysis of variance (ANOVA) followed by a post hoc LSD test (homogeneity of variance) and Tukey's test (heterogeneity of variance) using SPSS software (SPSS Inc., Chicago, USA). e significance level was defined as p < 0.05.

LIG Maintains NP Cell Vacuoles and IVD Score in LSI
Mice.
e effect of LIG on NP cells in vivo was assessed by using the LSI mice model. LSI surgery induced significant IDD as shown by IVD score after 4 weeks. LIG treatment increased IVD score compared with vehicle (Veh) treatment (Figures 1(a) and 1(b)). 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.

LIG Decreases TGFβ Activation in NP Cells of LSI Mice.
Vacuole sizes of NP cells are regulated by TGFβ activation. To investigate the effect of LIG on TGFβ activation during LSI-induced IDD, we examine the activation of TGFβ with the immunochemical assay. It showed latent TGFβ was activated after LSI surgery, indicated by significantly increased phosphorylated Smad2/3-positive (pSmad2/3+) cells in NP and annulus fibrosus (AF) (Figures 2(a)-2(c)). Decreased pSmad2/3+ cells were observed in the LIG treatment group relative to those in the Veh treatment group (Figures 2(a)-2(c)).

LIG Reduces TGFβ1 Expression in NP Cells of UP Rats.
To test the effect of LIG on NP cells in different animal models, we employed a rat model in which IDD is induced by upright posture [10]. We found that TGFβ expressions were significantly increased in NP cells of the UP + Veh group relative to the Sham group (Figures 3(a) and 3(b)). Continuous LIG treatment for 1 month significantly decreased TGFβ expressions in NP cells relative to Veh treatment (Figures 3(a) and 3(b)). activation are dose-dependent, we applied different doses of LIG on TGFβ pretreated IVD ex vivo. After 24 h, the protein levels of pSmad2 were significantly increased by TGFβ induction. e combination of LIG treatment decreased the levels of pSmad2 in a dose-dependent pattern. e dose of 10 − 5 M LIG exerted the most significant inhibitory effect on pSmad2 levels that were stimulated by exogenous TGFβ (Figure 4). is dose is chosen for further in vitro experiment.

Discussion
Nucleus pulposus cells, a remnant of embryonic notochord, are a unique cell type present in compartment of IVDs [17]. Overactivation of TGFβ is a key molecular event that mediates NP cell transition, leading to IDD development. Recently, new therapies have been reported to prevent IDD via targeting NP cells and extracellular matrix remodeling such as microRNAbased therapy and melatonin [18,19]. However, the therapy targeting NP cells through TGFβ activity has not been reported.
Our study revealed that LIG prevented NP cell transition and maintained vacuole sizes of NP cells. LIG was reported to exert chondroprotective effect by inhibiting chondrocytes apoptosis [20], attenuating the interleukin 1β (IL-1β)-induced Glycosaminoglycan (GAG) degradation and matrix metalloproteinase-3 (MMP-3) expression, increasing cell viability through suppression of ROS production, maintaining mitochondrial membrane potential, and downregulating caspase-3 activity [21]. It is also a potent blocker of vasoconstriction and a strong scavenger of oxygen-free radicals. LIG enhances vascularization by increasing vessel volume, vessel surface, and vessel thickness, upregulating vascular endothelial growth factor (VEGF) in femoral heads [22]. However, LIG is barely able to increase vascularization on NP cells since NP cells are avascular.
We found the effect of LIG on NP cells is associated with inhibition of TGFβ overactivation and TGFβ1 expression, which is similar to several reports on other organs or cell types. For example, LIG decreases the expression of TGFβ1 and reduces deposition of type III collagen during airway remodeling [23]. It also resists renal interstitial fibrosis by downregulation of TGFβ1 and CCN2 and upregulation of Smad7 [24,25]. LIG prevents and alleviates the development of liver fibrosis by downregulating TGFβ1, TβRII, CCN2, and Smad2/3, and type I collagen while upregulating Smad7 [26,27] and inhibits proliferation of hepatic stellate cells [28]. LIG prevents postoperative intra-abdominal adhesions by inhibition of pSmad2/3 expression and increasing the level of Smad7 in the peritoneal mesothelial cells [29]. Collectively, LIG prevents IDD via modulating TGFβ signal. ere are also some limitations in our study. We observed the effect of LIG on animal models for one month which is relatively short. Long-term effect has not been assessed in this study. e potential downsides of long-term treatment may include antifibrotic effect on tissue repair such as scar tissue formation.

Conclusion
LIG prevents NP cell transition by suppression of TGFβ overactivation. It can be developed as a therapeutic alternative for IDD.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.