Tuina inhibits ferroptosis and inflammatory response in nucleus pulposus of intervertebral disc degeneration rats

Abstract

Background

Intervertebral disc degeneration (IVDD) is a prevalent musculoskeletal condition that affects the spine, particularly in the lumbar region and induces low back pain. Tuina therapy has been widely utilized to alleviate low back pain for a long time. However, the specific mechanism underlying this effect of Tuina therapy on IVDD remains unclear.

Methods

The rat IVDD model was established by puncturing caudal vertebrae. Magnetic resonance imaging, Hematoxylin and eosin staining, and Safranin O staining were operated to assess histological changes. RT-qPCR and Western blot were carried out to measure mRNA and protein levels, respectively. Prussian Blue Staining and an iron assay kit were utilized to measure iron content in NP tissues.

Results

The chaotic fibrous tissues, loss of NP tissues and cartilaginous components were observed in IVDD rats, and these pathological changes were alleviated by Tuina therapy. Besides, the mRNA and protein levels of inflammatory factors (TNF-alpha, IL-1beta, and IL-6) were significantly upregulated in IVDD group compared to sham group, and Tuina therapy reduced the release of these inflammatory factors. Moreover, the increased content of iron ion in IVDD rats was also reduced by Tuina therapy. Furthermore, the upregulation of ACSL4 protein, the downregulation of GPX and FTH proteins in IVDD groups were reversed by Tuina therapy.

Conclusions

Tuina therapy prevented IVDD aggravation by suppressing inflammatory response and ferroptosis in rat model.

Keywords

Tuina IVDD ferroptosis inflammatory response

Introduction

Low back pain (LBP) is a prevalent issue in the general population, with 84% likely to experience it at some point in their lives.^1,2 Intervertebral disc degeneration (IVDD) is a major contributor to LBP, accounting for over 40% of cases and imposing significant socio-economic burdens worldwide.³ The intervertebral disc consists of the nucleus pulposus (NP) tissue, annulus fibrosus (AF) tissue, and endplates (EP) tissue.⁴ Various factors such as aging and biomechanical overload can lead to the fibrosis of NP tissue, decreased extracellular matrix (ECM) levels, ultimately causing IVDD.^5–7 Conservative treatment may result in drug dependence with inadequate pain relief, while surgical treatment is highly invasive with long recovery times and high recurrence rates.^8,9 Therefore, more effective and safer strategies are urgently needed for IVDD treatment.

Manual therapy or massage has been widely utilized as complementary and alternative therapy,^10,11 which involves hands-on manipulation of external areas of the body, with potential effects on internal physiological processes.^12,13 As a traditional form of manual therapy in China, Tuina encompasses pinching, pressing, and kneading techniques.^14,15 Moreover, the efficacy of Tuina in clinical practice for treating LBP or lumbar disc herniation has been confirmed.^16,17 Particularly, massage was identified to protect against oxidative stress injury by targeting JAK2/STAT3 signaling during IVDD trauma progression.¹⁸ However, there is limited research on the impact of Tuina on tissue degeneration beyond pain management, despite evidence linking LBP to age-related intervertebral disc degeneration.^13,19 A previous study indicated that Tuina can stimulate myofibroblast protein synthesis and function as a potential therapeutic intervention for age-related muscle injury, suggesting its potential to mitigate aging-induced tissue degeneration.²⁰ Furthermore, Tuina was reported to mitigate oxidative stress, potentially contributing to its analgesic effects.^21,22 Besides, Tuina was proven to attenuate chondrocyte apoptosis in IVDD rabbits by activating the FAK/PI3K/Akt pathway.²³ However, the potential mechanism of Tuina in IVDD deserves a further exploration.

Various pathological changes, including inflammation, ECM degradation, cell apoptosis, and cell death are considered as the primary causes of IVDD.^24–26 The maintenance of physiological function in the intervertebral disc during IVDD is contingent upon the delicate balance of ECM metabolism.⁸ Intervertebral disc cells produced or released several proinflammatory factors that initiate a cascade of pathological reactions, which further contributed to intervertebral disc cell autophagy, apoptosis or death.^27,28 Furthermore, these cytokines recruit immune cells to activate inflammation and promote disc degeneration.²⁸ Ferroptosis is a new form of programmed cell death, which is identified in recent studies.^29,30 Ferroptosis is dependent on iron and reactive oxygen species (ROS).³⁰ Processes involved include iron accumulation, ROS formation, lipid peroxidation, and glutathione depletion.^30,31 Besides, Glycolysis-derived lactate was reported to promote ACSL4 expression and histone lactylation to drive ferroptosis in IVDD.³² The Piezo1 ion channel exacerbates ferroptosis in nucleus pulposus cells by mediating mechanical stress–induced iron influx in IVDD.³³ Nevertheless, the relationship between Tuina and ferroptosis remains uncharacterized in IVDD.

In the current study, we attended to further explore the function and mechanism of Tuina in IVDD rats. Based on published literatures, we hypothesized that Tuina prevented IVDD aggravation by suppressing inflammatory response and ferroptosis in rat models. Our study may provide a novel insight into IVDD therapy.

Materials and methods

Animals

Total 50 adult Sprague–Dawley rats (male, 7 weeks, 220-240 g) were purchased from Shanghai Jihui Laboratory Animal Co., Ltd. (Shanghai, China) and housed in in standard laboratory conditions (temperature: 23 ± 2°C, humidity: 50%–70%, and a 12-h light-dark cycle) with rat chow and water ad libitum. After acclimatization for 1 week, rats were randomly divided into 5 groups (including sham + control, sham + Tuina, IVDD + control, IVDD + Tuina, and IVDD + Fer-1) based on a random number table. All animal operations were performed in accordance with procedures approved by the Experimental Animal Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd (HLK-20240305021-003; April 15, 2024). Experimental results were analyzed by researchers who are blind to animal groups.

IVDD model establishment

Rats were anesthetized using isoflurane inhalation. Once fully sedated, rats were placed in a prone position on the surgical table, and their tails were immobilized. The puncture site at the caudal vertebrae (Co7/8) was confirmed, and the annulus fibrosus of the intervertebral disc was penetrated using a 21-gauge needle. In detail, the needle was inserted parallel to the vertebral body and maintained a depth of 5 mm. Then, the needle was rotated 360° and maintained in position for 30 s before being slowly withdrawn along the original trajectory of insertion. The sham-operated rats received similar operations without puncturing.

Tuina therapy and ferrostatin-1 treatment

Tuina therapy was performed from day 2 after the surgery. The rats were placed in a massage manipulation finger-cot device for 30-min acclimatization before the intervention. Then the thumb and the index finger were used to press and knead the Shenshu (BL23) acupoints (located adjacent to the second lumbar vertebra) for 10 min once a day for 4 weeks. A tactile sensor system (FingerTPS, Pressure Profile System, Los Angeles, CA) was employed to monitor the intensity of massage operation, with a pressure of 5 N and a frequency of 120 times/min. Rats that received gently touch (almost no pressure) of Shenshu (BL23) acupoints served as control. Ferrostatin-1 was purchased from MedChemExpress (Shanghai, China. Purity: 99.71%) and intraperitoneally injected into rats (10 mg/kg) 24 h before model establishment.

Magnetic resonance imaging analysis

Four weeks post-Tuina therapy, the rats were euthanized, and their caudal vertebrae were collected for MRI imaging using a uMR 770 MRI system (United Imaging, Shanghai, China) to assess disc degeneration grades based on the Pfirrmann classification as previously described.³⁴

Histological analysis

The intervertebral disc tissues were harvested and fixed in a 4% paraformaldehyde (Solarbio, China) fixative solution for 48 h. Then, tissues were decalcified in 10% EDTA solution (pH 7.2–7.4) for 2 weeks, with daily solution changes. Subsequently, the harvested tissues were dehydrated in graded ethanol baths and embedded in paraffin wax. The paraffin-embedded tissues were sliced into sections (5µm-thick) by using a microtome. After rehydration and de-paraffinization, sections were subjected to hematoxylin and eosin (H&E) staining (Solarbio, China) to visualize the histological morphology of the intervertebral disc, while Safranin O staining (Solarbio, China) was used to observe cartilaginous components within the disc. Histological scoring of the H&E and Safranin O-stained samples was performed as described previously.³⁵ Total five perspectives were evaluated, and each category was assigned a score ranging from 1 to 3. Higher scoring levels indicated severer degrees of degeneration.³⁶

Real-time reverse-transcription polymerase chain reaction (RT-qPCR)

Total RNA was isolated from the NP tissues using TRIzol reagent (Takara Bio, Japan). The extracted RNA was reverse-transcribed into complementary DNA (cDNA) with a reverse transcription kit (Toyobo, Japan) following the manufacturer’s instructions. Subsequently, the synthesized cDNA underwent gene amplification via Polymerase Chain Reaction (PCR). The PCR system was prepared using SYBR Green-PCR Master Mix (Toyobo, Japan) as the dye, and Real-time PCR reaction was carried out according to the manufacturer’s protocol. The relative mRNA levels were determined using the 2-ΔΔCT method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as an internal control gene. Primer sequences can be found in Table1.

Table 1.

Sequences of primers used for reverse transcription-quantitative PCR.

Gene	Sequence (5’→3’)
Rat TNF-α forward	GGTGCCTATGTCTCAGCCTCTT
Rat TNF-α reverse	GCCATAGAACTGATGAGAGGGAG
Rat IL-1β forward	TGGACCTTCCAGGATGAGGACA
Rat IL-1β reverse	GTTCATCTCGGAGCCTGTAGTG
Rat IL-6 forward	TACCACTTCACAAGTCGGAGGC
Rat IL-6 reverse	CTGCAAGTGCATCATCGTTGTTC
Rat GAPDH forward	CATCACTGCCACCCAGAAGACTG
Rat GAPDH reverse	ATGCCAGTGAGCTTCCCGTTCAG

Western blot

The total protein was extracted from NP tissues using an extraction kit (Beyotime Biotechnology, Shanghai, China). Protein concentration was determined with the BCA protein assay kit (Beyotime Biotechnology). Subsequently, 20 μg tissue lysates were separated by 12% SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were then incubated overnight at 4°C with several primary antibodies after being coated with 5% BSA at room temperature for 2 h. Following this, the membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:5000, Sanggon Biotech, China) at room temperature for 2 h. Finally, the protein bands were acquired using the ChemiDoc MP system (Bio-Rad, USA) with an enhanced chemiluminescence (ECL) kit (Millipore, USA) and quantified using ImagePro plus 6.0. The information about primary antibodies was available in Table 2.

Table 2.

Primary antibody information.

Antibody	Dilution	Number	Source
TNF-α	1: 1000	ab183218	Abcam
IL-1β	1: 1000	ab283818	Abcam
IL-6	1: 1000	ab9324	Abcam
GPX4	1: 1000	ab125066	Abcam
FTH	1: 1000	ab183781	Abcam
ACSL4	1: 1000	ab155282	Abcam
GAPDH	1: 1000	ab8245	Abcam

Prussian Blue Staining

The paraffin-embedded sections were dewaxed and used for Prussian Blue Staining by using the Prussian Blue Staining kit (G1422; Solarbio, Beijing, China). The stained sections were then visualized and captured under an inverted microscope (DMI6000 B; Leica Microsystems Inc., Buffalo Grove, IL) as previously reported.³⁷

Iron concentration measurement

An iron assay kit (ab83366; Abcam, shanghai, China) was used to measure iron concentration. NP tissues were lysed in iron assay buffer and centrifuged (16,000◊g) for 10 min. The iron-reducing agent (5 mL) was added to the sample (50 mL) for total iron determination. Next, the iron probe solution (100 mL) was added to the sample and incubated at 4°C in darkness for 1 h. Absorbance at 593 nm was tested by a microplate reader (Thermo Fisher Scientific, Waltham, MA).

Statistical analysis

Data are presented as the mean ± standard deviation and analyzed using GraphPad Prism 8.0 software (GraphPad, San Diego, CA). Difference comparisons were conducted by one-way ANOVA followed by Tukey’s post hoc analysis. P < 0.05 indicated statistical significance.

Result

Tuina therapy alleviated disc degeneration of rats

We established an IVDD rat model to evaluate the protective effect of Tuina therapy on disc degeneration. According to T2WI MRI, the intervertebral space height and disc signal intensity were lower in IVDD group than those in sham group, and Tuina therapy increased the intervertebral space height and disc signal intensity (Figure 1(a)). Besides, the Pfirrmann grades were increased in IVDD group compared to sham group, and Tuina therapy reversed this result (Figure 1(b)). Collectively, Tuina therapy alleviated disc degeneration of IVDD rats.

Figure 1.

Tuina therapy alleviated disc degeneration of rats. (A) The representative images of MRI (n = 10/group). (B) The analysis of disc degeneration grades according to the MRI images (n = 5/group). ^***P < 0.001 compared to sham + control group. ^###P < 0.001 compared to IVDD + control group.

Tuina therapy improved histological injury in IVDD rats

Then, the discs with adjacent vertebral bodies were harvested for histological analysis. In H&E staining, the degenerated features IVD structure, the loss of NP and chaotic fibrous tissues were observed in IVDD group, and normal structure of intervertebral disc was observed in sham group. After Tuina therapy, the morphological changes in IVDD rats were partially ameliorated, and increased histological score in IVDD group was reduced by Tuina therapy (Figure 2(a)–(b)). According to Safranin O/Fast Green staining, the loss of cartilaginous components was observed in the intervertebral disc tissues from IVDD rats, and intact and normal cartilaginous components were observed in sham group. Moreover, Tuina therapy effectively inhibited the loss of cartilaginous components and histological score in IVDD group (Figure 2(c)–(d)). Taken together, Tuina therapy ameliorated histological injury in IVDD rats.

Figure 2.

Tuina therapy improved histological injury in IVDD rats. (A-B) The representative images of H&E staining of intervertebral disc and the quantification of histological score (n = 5/group). (C-D) The representative images of Safranin O staining of intervertebral disc and the quantification of histological score. ^***P < 0.001 compared to sham + control group (n = 5/group). ^###P < 0.001 compared to IVDD + control group.

Tuina therapy inhibited inflammatory response in vivo

To evaluate the effect of Tuina therapy on inflammation, the cytokines were detected by RT-qPCR and Western blot analyses. As depicted in Figure 3(a)–(c), the mRNA level of TNF-α, IL-1β, and IL-6 was increased in NP tissues from IVDD rats, and Tuina therapy inhibited the increase of cytokines. In addition, according to Western blot analysis, the upregulation of TNF-α, IL-1β, and IL-6 proteins in IVDD group was neutralized by Tuina therapy (Figure 3(d)–(g)), implying the anti-inflammation effect of Tuina therapy on IVDD.

Figure 3.

Tuina therapy inhibited inflammatory response in vivo. (A-C) The measurement of TNF-α, IL-1β, and IL-6 mRNA levels in NP tissues (n = 5/group). (D-F) The detection of TNF-α, IL-1β, and IL-6 protein levels in NP tissues (n = 5/group). ^***P < 0.001 compared to sham + control group. ^##P < 0.01, ^###P < 0.001 compared to IVDD + control group.

Tuina therapy attenuated ferroptosis in vivo

We then evaluated the effect of Tuina therapy on ferroptosis. According to Prussian blue staining, the Fe³⁺ content in NP tissue was higher in IVDD groups than that in sham group, which was further reduced by Tuina therapy or Fer-1 treatment (Figure 4(a)). Additionally, the increased concentration of iron salt in IVDD group was reversed by Tuina therapy or Fer-1 treatment (Figure 4(b)). Moreover, Western blot analysis demonstrated that the upregulation of ACSL4 protein, the downregulation of GPX and FTH proteins in IVDD groups were counteracted by Tuina therapy or Fer-1 treatment (Figure 4(c)–(f)). In summary, Tuina therapy or Fer-1 treatment attenuated ferroptosis in IVDD rats.

Figure 4.

Tuina therapy attenuated ferroptosis in vivo. (A) The representative images of Prussian blue staining of NP tissues (n = 5/group). (B) The measurement of iron salt in NP tissues (n = 5/group). (C-F) The detection of ACSL4, GPX and FTH protein levels in NP tissues (n = 5/group). ^***P < 0.001 compared to sham + control group. ^###P < 0.001 compared to IVDD + control group.

Discussion

Endplate dysfunction is considered a significant factor in the development of IVDD, which is the primary source of nutrition and cytokine production.³⁸ The degenerate disc exhibits a decrease in endplate permeability to 50-60% and marked inhibition of solute transport.³⁹ Furthermore, calcification of the endplate impeded the transport of nutrient and oxygen, contributing to IVDD progression.⁴⁰ To establish IVDD model, a needle was punctured parallel to the endplate to avoid disruption in our study. Notably, endplate injury was observed in the IVDD group as evident by decreased disc signal intensity according to MRI imaging and histological staining, and MRI images revealed blackening at the border of the endplate in IVDD rats. However, this injury was ameliorated by Tuina therapy.

ECMs within NP tissues are critical constituents in the intervertebral disc, as it is responsible for maintaining hydration and structural integrity.²⁵ Dysregulation of ECMs contributes to disc degeneration, which is characterized by the loss of these essential components.^41,42 Previously, Tuina exerted protective effect on intervertebral disc tissue morphology in rabbit with IVDD.²³ Besides, Tuina therapy improved degenerative structural defects and inhibited cell arrangement disorders in aging rats.⁴³ Similarly, our study demonstrated that Tuina alleviated histological injury by reducing the loss of NP tissues and cartilaginous components and reversing chaotic fibrous tissues.

Emerging publishers proposed that the release of inflammatory factors on the NPCs contribute to the injury or degeneration of the IVD.^44,45 The inhibition of cytokine production can be utilized in the treatment of IVDD, as it has the potential to delay this process and alleviate the pain associated with IVDD.^45,46 For instance, digoxin suppressed the release of inflammatory factors via inactivation of TNF/NF-κB signaling in IVDD rats.³⁵ Consistent with previous research, our study suggested that Tuina therapy not only decreased mRNA level of cytokines but also downregulated protein level of inflammatory factors in NP tissues, implying the anti-inflammatory effect of Tuina therapy.

Ferroptosis is primarily characterized by intracellular iron ion or ester oxygenase-induced catalysis of unsaturated fatty acids on cell membranes, leading to lipid peroxidation and oxidative injury.⁴⁷ This process is further distinguished by the depletion and inactivation of glutathione system.²⁹ The anti-ferroptosis effects are mediated by glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH), which respectively catalyze lipid hydroperoxides and transport intracellular iron ions.⁴⁸ Additionally, long-chain acyl‐CoA synthetase 4 (ASL4) has been implicated in the initiation or activation of ferroptosis, demonstrating a positive association with this cellular process.³⁰ Inhibition of ferroptosis has widely reported to alleviated IVDD effectively.⁴⁹ For instance, deubiquitinase USP11 was proven to inhibit oxidative stress-induced ferroptosis by promoting the deubiquitylation and stabilization of Sirt3 in IVDD rats.⁴⁸ Besides, Cynarin protected NP cells from ferroptosis and further alleviated IVDD progression.⁵⁰ In our study, increased Fe³⁺ and iron salt contents in NP tissues were observed in IVDD rats, and Tuina therapy or Fer-1 treatment effectively reduced Fe³⁺ and iron salt contents. Moreover, the upregulation of ACSL4 protein, the downregulation of GPX and FTH proteins in IVDD groups were reversed by Tuina therapy or Fer-1 treatment. All these results confirmed the inhibitive effect of Tuina therapy on ferroptosis in IVDD rats.

Nevertheless, there are several limitations in our study. The current study focused on the effect of Tuina on ferroptosis, while other biological processes such as inflammatory response or mechanical loading on ferroptosis were ignored. Besides, the current study is the primary study of Tuina therapy on IVDD. There is still a long way to go before the clinical trials of this therapy. At last, the potential specific mechanism of Tuina therapy affecting inflammation or ferroptosis remains obscure.

In conclusion, we demonstrated that Tuina inhibited the ferroptosis and inflammatory response in nucleus pulposus of intervertebral disc degeneration rats. This offers a novel avenue for future investigation into the molecular mechanisms involved in IVDD and its therapeutic interventions.

Supplemental material

Supplemental material - Tuina inhibits ferroptosis and inflammatory response in nucleus pulposus of intervertebral disc degeneration rats

Supplemental material for Tuina inhibits ferroptosis and inflammatory response in nucleus pulposus of intervertebral disc degeneration rats by Qian Li, Gangfeng Duan, Junchao Xu, Li Chen, Zhonghui Chen in Journal of Orthopaedic Surgery

Footnotes

Acknowledgement

We appreciate the support of Mongolian Medical Hospital of Bortala Mongolian Autonomous Prefecture.

ORCID iD

Zhonghui Chen

Ethical considerations

All animal operations were performed in accordance with procedures approved by the Experimental Animal Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd (HLK-20240305021-003; April 15, 2024).

Authors’ Contributions

Qian Li and Gangfeng Duan conceived and designed the experiments. Qian Li, Gangfeng Duan, Junchao Xu, Li Chen and Zhonghui Chen carried out the experiments. Qian Li, Gangfeng Duan, Junchao Xu, Li Chen and Zhonghui Chen analyzed the data. Qian Li, Gangfeng Duan and Zhonghui Chen drafted the manuscript. All authors agreed to be accountable for all aspects of the work. All authors have read and approved the final manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work was supported by Science and Technology Bureau Project of Bortala Mongolian Autonomous Prefecture (Massage of Bushen Tongdu Meridian Points on Pre-intervertebral Disc Degeneration Based on GPX4 Mediated Iron Death, Zdkj2023001).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.*

Supplemental material

Supplemental material for this article is available online.

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