Whole-body Vibration Attenuates Pyroptosis-Mediated Inflammation but Accelerates Progression of Intervertebral Disc Degeneration

Whole body vibration (WBV) is a non-pharmaceutical therapy that has been widely incorporated into clinical practice for musculoskeletal disorders, including low back pain (LBP). Intervertebral disc (IVD) degeneration (IVDD) is clinically associated with LBP and is known as the main cause for LBP. However, cumulative evidence also suggested WBV might have an adverse impact on IVDs. Moreover, previous studies have been focusing on the effects of WBV on healthy mice, rather than those suffering from IVDD. Thus, uncertainties still exist concerning the effects of WBV on IVDs undergoing IVDD. This study was aiming to evaluate the effects of WBV intervention on the development and progression of IVDD mouse model induced by lumbar spine instability (LSI) surgery. This scale mainly scores the disruption of nucleus pulposus central cavity, cellularity and collagen fiber orientation of annulus fibrosus and the degree of ectopia ossification of cartilage endplate. Simple radial clefting = the presence of radial gaps between AF lamellae with minimal fragmentation; complex radial clefting = the presence of radial, transverse, and/or oblique gaps in the lamellae with significant fragmentation; junction = the triangle junction between the cartilage endplate and the annulus fibrosus.

prevalence of LBP in workers exposed to vibration [15]. Furthermore, increasing the majority of basic studies believed that WBV may be detrimental to the morphology of joint itself [16,17]. Nevertheless, investigation on the bone effects of different WBV frequencies demonstrated that WBV induces local effects in bone that differ based on the anatomic site (e.g., vertebrae, femur or tibia) [18]. And the majority of basic studies have focused on the effects of WBV on normal IVDs, rather than those suffering from back pain provoked by IVDD. Therefore, the widespread use of WBV still requires more efficacious and rigorous research-based evidence for addressing the effects of WBV on IVDs undergoing IVDD or LBP.
Previous studies have proved that Wnt/β-catenin signaling pathway is activated and involved in matrix degradation, apoptosis of IVD cells and following IVDD progression [19,20]. In this study, we aimed to evaluate the roles of WBV intervention on the pathological and biochemical changes of an IVDD mouse model, C57BL/6J mice first received LSI surgery to induce IVDD model and further exposed to a commercial WBV platform for 8 weeks. The lumbar vertebrae were harvested to evaluate the effects of WBV on changes of water content, height, tissue morphology, matrix metabolism, distribution of sensory nerves of IVD, apoptosis and pyroptosis of IVD cells as well as the activity of the Wnt/βcatenin signaling. The results may raise our understanding regarding the effects of WBV on the degeneration of lumbar IVD of humans.

Micro Computed Tomography (μCT)
The lower thoracic and whole lumbar spine from mice were dissected, fixed in 4% buffered paraformaldehyde for 72 h, transferred into phosphate-buffered saline, and then examined by high-resolution µCT (Skyscan 1176; Bruker µCT, Kontich, Belgium). The ribs on the lower thoracic were included for the identification of L4-L5 IVD localization. Images were reconstructed and analyzed using NRecon v1.6 and CTAn v1.9 (Skyscan company, San Jose, CA, USA), respectively. Three-dimension model visualization software, CTVol v2.0 (Skyscan company, San Jose, CA, USA), was used to analyze parameters of the L4-L5 IVD with the half-height of the L4 and L5 vertebrae. The scanner was set at a voltage of 90 kV, a current of 300 µA, and a resolution of 9 µm per pixel to measure the IVD. A resolution 9 µm per pixel was set for the whole L5 vertebral body measurement. Coronal images of the L4-L5 IVD were used to perform three-dimension histo-morphometric analyses of IVD. IVD volume was defined by the region of interest to cover the whole invisible space between L 4 and L 5 vertebrae. were normalized against β-actin. The relative expression levels were calculated using the 2 -ΔΔ CT method [24].

Statistics Analysis
All the data were expressed as mean ± s.d., as indicated in figure legends. All data analyses were performed using SPSS 15.0 analysis software (SPSS Inc, Illinois, USA).
Statistical differences between groups were determined using one-way analysis of variance (ANOVA) or Student's t-test. and the level of significance was defined as P < 0.05.

WBV Treatment Aggravates the Progression of IVDD Induced by LSI Surgery
To determine the effect of WBV on the progression of IVDD, mice in the IVDD group and WBV group received an LSI surgery to induce IVDD model as previously described [25], and then mice in the WBV group received multiple repeated whole-body vibration for 8 weeks (Fig. 1b-c). MRI scan results showed that LSI surgery was successful in producing IVDD modeling, resulting in remarkable signal loss of T 2 -weighted image in IVD of IVDD mice, whereas virtually no signal loss in that of the SHAM mice (Fig. 2a). Compared to the IVDD group, WBV treatment further aggravate the decline of signal intensity (Fig. 2b). The IVD height of mice generally increased from birth to 1-month old, and then remained stable before decreasing around 4-month old [25]. Since the reduction of IVD height is commonly regarded as a surrogate predictor of IVDD, we evaluated the disc height index (DHI) between the L4-L5 by µCT analysis. And we found DHI in IVD mice is gradually decreased and a significant difference of DHI was observed at 8-week post-surgery, compared with SHAM mice. Unfortunately, this notable decreased DHI was already exhibited in WBV mice 2-week post-surgery ( Fig. 2c-d). Moreover, no other obvious difference in BS/TV, Tb.N and Tb.Sp, was observed between the IVDD mice and WBV mice except for a notable decrease in Tb.Sp in WBV mice (Fig. 2d). And we speculate that the decrease of trabecular space may result from the increase of bone mass by WBV treatment as previously described [11,26].
Histological analysis of L4-L5 IVD by Safranin O/Fast green staining subsequently showed that the SHAM mice had well-organized IVD structures, including large vacuoles with sufficient matrix content in NP, arranged regularly AF tissues without any tearing, and no ectopic bone formation in EP, whereas WBV aggravated the IVDD phenotype induced by LSI surgery, including more severity of fissures and folds in the interlamellar of AF tissues, reduction in height and vacuole sizes in NP, formation of ectopic bone in EP (Fig. 3a). The deterioration of IVDD was further assessed by the histological score. As shown in Fig. 3b, NP, AF and EP scores were decreased in LSI-treated mice and WBV-treated mice compared with SHAM-treated mice, and a significant decrease of AF score exhibited in IVDD-mice since 2-week post-surgery (P < 0.05), and appeared earlier in WBV mice since 1-week post-surgery (Fig. 3b). All these radiological and morphologic results proved that WBV has deleterious effects on LSI-induced IVDD mice.

WBV Stimulates Apoptosis Of IVD Cells Of IVDD Mice
Besides, apoptosis of IVD cells, a process of cellular suicide, has been found to participate in the degeneration of IVD [22,27,28]. We next investigated the effects of WBV on apoptosis of IVD cells by TUNEL assay. Consistent with previous results, LSI-surgery strongly induced apoptosis of IVD cells in IVDD mice, and this increase of apoptosis in IVD cells was further amplified after WBV intervention (Fig. 3c).

WBV Intensifies Matrix Degradation In IVDD Mice
We determined the effects of WBV on matrix degradation in IVD. As the most abundant week post-surgery and at 8-week post-surgery, respectively (Fig. 3c-f). These results suggested that WBV accelerates IVDD progression by increasing the matrix degradation of IVD tissue.

WBV Increases Innervation and Sensory Nerve Ingrowth into NP region of IVDD Mice
IVDD is thought to lead to LBP because of nerve ingrowth into the degenerate IVD [5].
Evidence from animal models and humans has revealed sensory innervation of lumbar IVD and sensory nerve ingrowth into the inner layer of IVD, causing painful conditions [29][30][31].
To explore the distribution of constant nociceptive sensory nerves after WBV treatment, ingrowth into the NP region of IVDD mice (Fig. 5a-b).

WBV Attenuates Caspase-1 And IL-1β Expression In IVDD Mice
Studies have found that a cause of discogenic LBP is IVD inflammation and axonal growth of afferent fibers innervating the disc, and IL-1β is a pain-related molecule that was significantly elevated in painful human IVD [32]. We have previously reported that Nlrp3 inflammasome-mediated pyroptosis is proinflammatory and produces an excessive level of cartilage-degrading cytokines, including IL-1β [33]. To date, the relationship between pyroptosis and IVDD is still obscure. We speculate pyroptosis may also participate in the progression of IVDD and WBV intervention. Thus, we examined the expression changes of IL-1β, Nlrp3 and Caspase-1 (two key proteins in pyroptosis). Unexpectedly, IHC results of Caspase-1 and IL-1β demonstrated that LSI-treated mice had significantly increased Caspase-1 and IL-1β levels in NP and AF, whereas WBV-treated mice exhibited decreased Caspase-1 and IL-1β in NP and AF (P < 0.01) (Fig. 6c-f). However, almost no difference in expression of Nlrp3 protein between IVDD mice and WBV mice was observed except a slight increase in Nlrp3 at 4-week post-surgery (P < 0.05) (Supplementary Fig. 1b, c).
These findings indicated that WBV may have a pain relief effect on IVDD mice, partially due to the decrease of IL-1β and pyroptosis in IVD tissue.

WBV Activates Wnt/β-catenin Signaling Pathway Of IVDD Mice
The previous studies have demonstrated that β-catenin protein is up-regulated and activated in disc tissues from patients with disc degeneration [27]. We investigated whether the activity of Wnt/β-catenin signaling is involved in WBV intervention mediated aggravation of IVDD. As expected, IHC results of β-catenin gradually increased at 2-week and peaked at 4-week post-surgery in IVDD mice, compared with SHAM mice. However, a higher peak appeared at 1-week post-surgery (2.8 folds of that in IVDD mice) following WBV treatment, thereafter reduced to 48% of that in IVDD mice at 2-week post-surgery ( Fig. 6a,b). We further determine the activity of Wnt/β-catenin signaling by analyzing the RNA expression levels of β-catenin and Lef1 (a target gene of Wnt/β-catenin signaling) in IVD tissues by qPCR analysis. Consistent with previous IHC results of β-catenin, β-catenin and Lef1 mRNA levels started to increase at 1-week and peaked at 2-week in IVDD mice, whereas WBV treatment advanced β-catenin and Lef1 mRNA levels to 1-week post-surgery, and gradually decreased later (Fig. 6c). These results suggest that LSI surgery leads to the activation of Wnt/β-catenin signaling, and WBV may accelerate and exacerbate the degeneration of IVDs through earlier and stronger activation of Wnt/β-catenin signaling.

Discussion
Given that WBV is being used clinically to treat musculoskeletal disorders, followup work should investigate the response of degenerative IVDs tissues to WBV, to better mimic and understand its clinical use in humans. Surprisingly, to our knowledge, little data has been attempted to determine the effect of WBV on morphological and biomechanical changes of animal models who already receiving IVDD model, whereas most previous studies have used healthy mice, and usually no degenerative changes have been found in IVDs [16,17].
Moreover, there is an urgent need to raise understanding of the mechanisms why patients with LBP reported great pain relief after WBV treatment. In this study, we found WBV indeed aggravates IVDD progression, including greater signal loss in T 2 -weighted image, advanced decrease in IVD height and AF scores (since 1-week after LSI surgery), larger numbers of apoptotic cells, acceleration of matrix catabolism, innervation into AF and ingrowth of sensory nerves into NP. In addition, WBV treatment results in earlier activation of Wnt/β-catenin signaling in IVDD mice.
Persistent inflammation in painful discs, and that the production of these molecules may be a major factor in discogenic LBP. Nlrp3-mediated pyroptosis is a new form of programmed inflammatory cell death that participates in the inflammatory response by releasing a plentiful of cartilage-degrading cytokines, including IL-1β [33]. After Nlrp3 binds its adaptor apoptosis-associated speck-like protein, pro-Caspase-1 will be recruited to assemble Nlrp3 inflammasome. After then, the active Caspase-1 was released from it and further to hydrolyzes pro-IL-1β into mature IL-1β [34]. Our previous finding was consistent with those of the latter study, which demonstrated Nlrp3 inflammasomemediated pyroptosis was induced during the progression of OA [33,34]. However, the role of pyroptosis during the progression of IVDD and WBV is not yet utterly understood. For the first time to our knowledge, we revealed that LSI-treatment significantly induces pyroptosis by increasing the protein levels of Nlrp3, Caspase-1 and IL-1β in IVDs.
Interestingly, WBV treatment significantly reduced the LSI-induced upregulation of Caspase-1 and IL-1β. This finding suggests that increase of pyroptosis and IL-1β in IVDs may account for increasing LBP for human suffering with IVDD, and inhibition of pyroptosis and IL-1β in IVDs could be one crucial mechanism for WBV in the relieving the LBP, which is parallel with the results stated by trial researches that patient self-reported pain relieve after WBV treatment in human.
Pathological innervation of the disc by pain-sensing nerve fibers is thought to be a key component of discogenic pain [35,36]. In this study, we found that IVDD mice exhibited a significant increase in the number of Cgrp + sensory nerves both in outer AF and ectopic bone formation zone, and WBV treatment further increases the density of Cgrp + sensory nerves in outer AF. These results further indicate that more nociceptive sensory innervation in AF could be an important origin of LBP in IVDD patients. Yet, the induction of nerve ingrowth into the disc was not explicitly investigated. Previous studies have reported not all degenerative discs are symptomatic, and vice-versa [37]. Base on our current results, we hypothesize that any general consideration of LBP should pay close attention to the discs, not only water content, disc height, viscoelastic behavior, strength of the vertebrae as indicated by water content (MRI), or bone density (CT) and even levels of the local cytokines (IL-1β) in IVDs, but also the number and localization of sensory nerves in IVDs. Despite trial researches have reported WBV could alleviate pain in human LBP caused by various spinal-related diseases [13,14], short duration of WBV treatment on LBP showed that the effects of WBV alleviating LBP seem to be temporary, when the vibration ceased, LBP returned to pre-vibration levels [38]. For these reasons, we speculate that pain relief requires long-term use of WBV to be sustainable and effective, the source of LBP cannot be eliminated fundamentally. Also, for LBP patients who have already started to use WBV, if they stop using WBV for a period of time, their LBP may be aggravated due to the innervation into IVDs induced by WBV intervention.
Interestingly, parallel with numerous work, our recent finding demonstrated that WBV could be beneficial to fracture healing in ovariectomized rats with bone fracture model, which indicated that WBV may be favorable to fractures healing in individuals with postmenopausal osteoporosis [26]. Of concern is the fact that the effects of WBV on joint tissues have not been thoroughly evaluated. There is a serious polarization in the understanding of WBV's effects on joint health [13][14][15][16][17]. It should be noted that there is currently no consensus on the relative importance of specific parameters of WBV (e.g., amplitude, frequency and duration of exposure) on bone adaptation, because recent trial reports showed contradictory results [39]. Based on previous studies that demonstrated beneficial effects of WBV on muscle and bone [12]. The parameters of WBV (3 Hz, and 0.4 g peak acceleration) used in the current study were selected to introduce the specific value of peak acceleration and frequency, which are comparable to those equipment used for human joint health and considered safe by ISO-2631 [12], and parameters of WBV also fall within the average range of vibrations produced in common environments [37].
Therefore, one limitation of this study is that the IVDD mice are only intervened with WBV of fixed frequency and amplitude for IVDD mice. Thus, our findings may be incomprehensive to evaluate the potential benefits for IVDs if exposed to even greater acceleration during WBV intervention. Further studies on the effects of WBV with a range of amplitudes and high frequencies corresponding to those used in the usual WBV vehicles on an ongoing IVDD model will give us more comprehensive insights.
Previous studies have proved that the Wnt/β-catenin signaling pathway is activated in a degenerative joint disease like OA [40][41][42][43]. In vitro studies have reported activation of Wnt/β-catenin signaling leads to induction of cell cycle arrest and apoptosis of IVD cells, increases of matrix catabolic degradation enzymes to accelerate matrix degradation, thereby promoting IVD degeneration [19,44,45]. However, the relationship between WBV and Wnt/β-catenin signaling in IVDD mice is largely elusive. Our results found that WBV not only contributes to earlier and stronger activation of Wnt/β-catenin signaling in IVDD mice but also augments LSI surgery-induced apoptosis of IVD cells and expression levels

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