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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 2 | Page : 160-166
Isobaric tag for relative and absolute quantitation-based proteomics for investigating the effect of Guasha on lumbar disc herniation in rats
Min Yang1, Gui-Hua Xu2
1 Office of Traditional Chinese Medicine Nursing Education and Research, School of Nursing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
2 Institute of Integrated Traditional Chinese and Western Medicine Nursing, Nanjing University of Chinese Medicine, Qixia District, Nanjing, Jiangsu, China
Correspondence Address:
Dr. Min Yang
Office of Traditional Chinese Medicine Nursing Education and Research, School of Nursing, Nanjing University of Chinese Medicine, No. 138, Xianlin Road, Qixia District, Nanjing, Jiangsu
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.370107
Objective: The objective of the study is to examine the possible mechanism by which Guasha (scraping therapy) affects the expression profiles of proteins in a lumbar disc herniation (LDH) rat model using isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics. Methods: Thirty-six rats were used in this study. LDH rats were subjected to noncompressive LDH surgeries. Rats were randomly divided into the model and Guasha groups. Guasha was applied on alternate days for a total of nine times (three courses). At the end of each course, six rats were randomly selected from each group and their blood samples were collected. iTRAQ labeling was used to examine the mechanism of action of Guasha against LDH. The molecular functions, cellular components, and biological processes were analyzed using gene ontology analysis. The Ingenuity Pathway Analysis database was used to identify canonical pathways involving these proteins. Results: Compared to the model group, 198, 182, and 170 proteins were identified as differentially expressed at the three respective Guasha treatment courses. Pathways, including focal adhesion kinase signaling, acute-phase response signaling, and the LXR/RXR activation pathway, were closely related to the effects of Guasha in LDH rats. Furthermore, Rac1, Orm1, and Hpx were validated by western blotting, and the results were consistent with the protein expression levels observed using the iTRAQ method. Conclusion: Guasha could not only regulate the pathological changes related to LDH, but also achieve therapeutic effects by stimulating physiological changes. Our results offer a better understanding of the effects of Guasha on LDH.
Keywords: Guasha, inflammation, isobaric tags for relative and absolute quantitation, lumbar disc herniation, proteomics
Lumbar disc herniation (LDH), a common health problem and cause of work disability,[1] is characterized by sciatica,[2] lower back pain,[3] and sensory or motor deficits.[4] LDH is an inflammatory condition, and the expression of inflammatory markers has been reported in LDH patients.[5] In addition, inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-alpha, and IL-6, are crucial in the development or continuation of neuropathic pain in LDH.[6],[7],[8] Currently, surgical and conservative interventions are the main treatments for LDH, although conservative therapy remains the basis of initial treatment in many patients diagnosed with LDH. Clinical and epidemiological studies have shown that most LDH patients can heal with conservative therapy.[9] Furthermore, there are strict restrictions on surgical indications; only a carefully selected number of LDH patients (ranging from 2% to 4%) are suitable for surgery.[10] Conservative therapy for symptoms associated with LDH includes pharmacologic and epidural steroid injections,[11] with NSAIDs, corticosteroids, narcotics, and muscle relaxants as conventional drugs.[12] However, pharmacological interventions may only be effective for a short time.[13] In addition, these drugs are associated with severe side effects such as gastrointestinal discomfort and liver or kidney toxicity.[14]
Given these limitations, traditional Chinese medicine (TCM), a noninvasive intervention with improved therapeutic effects, has been favored by patients and researchers over a broad geographic area.[15]Guasha (scraping therapy) is widely used for LDH as a “green” treatment. Bibliometric analysis has shown that Guasha can treat more than 400 types of disease, especially painful musculoskeletal problems.[16] Clinical and experimental studies have shown that Guasha can attenuate pain,[17] stimulate an unknown analgesic biochemical pathway,[18] and modulate cytokines.[19] Our research team previously found that the expression of inflammatory cytokines was significantly reduced in the serum of LDH rats after Guasha compared with the model group.[20],[21],[22] In addition, Guasha inhibited the T-helper 1 (Th1) type of immunity in LDH rats induced by autologous nucleus pulposus (NP), thereby promoting the Th1/Th2 balance and alleviating the pain caused by LDH.[23] However, the mechanism underlying this effect remains to be fully elucidated.
Proteomic research illustrates the different expressions of proteins in the blood, urine, and cells.[24] This allows the altered proteins to be identified as potential targets for therapy. Meanwhile, researchers may deduce the mechanism of intervention from the global analysis of proteins,[25] including but not restricted to the components, expression level, the modified situation of proteins, and the interactions and connections among them. Proteomics has high convergence with the theory of TCM, which is a holistic and systemic theory. Therefore, it provides a good opportunity to illuminate the mechanism of Guasha on LDH. Among proteomic tools, isobaric tags for relative and absolute quantitation (iTRAQ) have been widely used in protein identification and quantification owing to their high throughput, accuracy, and sensitivity.[26] Thus, the purpose of this study was to elucidate the mechanism of action of Guasha on LDH using iTRAQ-based proteomic analysis in rats with LDH with or without Guasha treatment.
MethodsExperimental animals
Thirty-six Sprague–Dawley rats, weighing 300–400 g each, were acquired from the Experimental Animal Center of Zhejiang Province (Zhejiang, China, Animal certificate: SCXK [Zhejiang] 2014-0001). The rats were housed in the specific pathogen-free laboratory animal center at Nanjing University of Chinese Medicine (Nanjing, China). They were fed ad libitum and were raised at 23.0°C ± 2.0°C with a 12-h light/dark cycle. After 1 week of acclimatization, rats were randomly divided into two groups: the LDH model group (model group, n = 18) and the LDH plus treatment with Guasha group (Guasha group, n = 18). Animal experiments were approved by the Ethics Review Committee of Nanjing University of TCM (ethical review permission number: 201910A018; date of approval of ethical review permission: November 15, 2019). The treatment of all rats complied with the Guidelines of Accommodation and Care for Animals established by the Chinese Convention for the Protection of Animals for Experimental and Other Scientific Purposes.
Establishment of the lumbar disc herniation rat model
As previously described,[27] a model of LDH induced by the contact between the NP and the diagnosis-related group (DRG) was established. In all surgical procedures, rats were anesthetized by injecting 10% chloral hydrate (0.35 mL/100 g). First, a midline incision was made in the dorsal and thoracolumbar fascia of the right L4–L5 spinous process. The paraspinous muscles were dissected, following which, to expose the right L5 DRG, partial unilateral laminotomy and medial facetectomy were conducted. The first coccygeal intervertebral disc was exposed bilaterally, the NP was taken through the horizontal cut in the annulus fibrosus, and the gel-like NP was promptly placed on the L5 DRG. Finally, the surgical wound was sutured in a single plane with the muscle fascia and skin. All animals were injected intraperitoneally with penicillin for 3 consecutive days postoperatively to avoid potential infections resulting from the modeling.
Guasha intervention
Rats in the Guasha group received Guasha treatment on the 5th day after surgery, performed as previously described.[20] The sequence of Guasha was as follows: first scraped the Governor Vessel on the back, then scraped the Bladder Meridian, and finally scraped the right hind limb of the rat. When scraping over the incision for the first time, the scraping force should be reduced appropriately. In addition, the bilateral acupoints Shenshu (BL 23), Weizhong (BL 40), Huantiao (GB 30), and Yanglingquan (GB 34) were scraped heavily. Guasha was repeated in one direction 10–20 times at every location until patterned ecchymosis emerged. Guasha was conducted on alternate days, three times per course, and each rat received between one and three courses. All rats were scraped by the first author, YM, who was fully trained to guarantee the equality of strength and times of Guasha before the actual practice, thus achieving consistency.
Behavioral observations
Rats in both groups were subjected to behavioral observations at a fixed time (10:00–12:00). Observers watched the rats to determine if symptoms of spinal cord injury, such as gait instability, foot valgus, irritability, and decreased appetite occurred. The evaluators were unaware of the grouping information.
Pain threshold determination
Thermal hyperalgesia of the rats was measured using a smart hotplate. The right hind paw of the rat was exposed to a thermal stimulus (52.0°C ± 0.2°C), and the latency to withdrawal evoked by the stimulus was recorded. The pain threshold was measured using paw withdrawal thermal latency (PWTL). The test was repeated three times, the average value was taken as the PWTL, and the interval between the two experiments was 15 min. The assessment was performed the day before modeling for the baseline and on the 4th day (the day before treatment).
Serum sampling, protein extraction quantification
On the 10th, 16th, and 22nd days after surgery, that is, after 1, 2, or 3 treatment courses, respectively, six rats were randomly selected from each group, and their blood was collected from the orbit. After an hour of rest at room temperature, these blood samples were centrifuged (3,000 rpm/20 min) and stored at −80°C separately for later use.
At each point in time, equivalent serum from each group was run on multiple affinity removal system columns (Agilent, Palo Alto, California, USA) to remove high-abundance proteins. A concentrated low-abundance protein solution was then obtained, and its concentration was detected using the Bradford method (Bio-Rad Protection Assay; Bio-Rad Hercules, California, USA).
Protein digestion and peptide isobaric tags for relative and absolute quantitation labeling
With the protein: trypsin ratio of 100:3.3, Trypsin Gold (Promega, USA) was applied to digest 100 μg of protein in each sample for 24 h at 37°C, after which trypsin was added once for 12 h at 37°C according to the above-mentioned ratio. In line with the manufacturer's instructions, the peptides were labeled with the iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex, U.K.). Peptides were labeled as follows: Model group, iTRAQ reagents 112, 113, and 114; Guasha group, iTRAQ reagents 115, 116, and 117. The iTRAQ-labeled peptides were separated using a strong cation exchange column.
Nano-liquid chromatography and mass spectrometry analysis
Nano-LC-mass spectrometry (MS)/MS analysis was conducted using an LTQ-Orbitrap (Thermo Fisher, CA) and a liquid chromatography system (Agilent 1,100 series) with a reversed-phase microcolumn (0.075 mm × 150 mm, Acclaim® PepMap100 C18 column, 3 μm, 100 Å; Dionex, Sunnyvale, CA, USA). The mobile phase consisted of buffer A (an aqueous solution containing 0.1% formic acid) and buffer B (a 95% acetonitrile solution containing 0.1% formic acid). The analytical environment was set as follows: linear-gradient buffer B (from 0% to 60% in 60 min) at a velocity of 200 nL/min. The column was rebalanced for 10 min in the initial state. MS scanning was performed using LTQ-Orbitrap Veces. The scan range of the grade I MS was 400–1,800 Da. For tandem MS, the scan range was 100–200 Da. After eight MS/MS scans of the eight peaks, a complete MS scan was required.
Data processing and analysis
Proteins were identified using the MASCOT search engine (http://www.matrixscience.com), based on the NCBInr and Swiss-Prot protein databases. Differential proteins were screened according to differences in expression between the groups. Fold changes of more than 1.3 or <0.77 and P < 0.05 (Student's t-test) were set as the thresholds for identifying significant changes.
Bioinformatic analysis
To clarify the functional classification of molecular functions (MFs), cellular components (CMs), and biological processes (BPs) of proteins regulated by Guasha in the serum of LDH rats, an analysis via gene ontology (GO) (http://www.geneontology.org/) was conducted. In addition, according to the rationality of the statistical algorithm and reliability of the database, Ingenuity Pathways Analysis (IPA, Ingenuity Systems, http://www.ingenuity.com/), which can map per adjusted protein to its corresponding genetic target in the Ingenuity Pathways Knowledge Base, was chosen to elucidate canonical pathway protein interactions.
Western blot analysis
Proteins (Rac1, Orm1, and Hpx) identified by iTRAQ were validated using western blotting. They were extracted at a low temperature and quantified and separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The proteins were then transferred electrically to polyvinylidene difluoride membranes. After blocking with 5% dried skim milk at room temperature for 90 min, the membranes were incubated with the primary antibodies (1:1,000) overnight at 4°C. They were then washed with TBST three times for 10 min each and incubated with the secondary antibodies (1:3,000) for 1 h at room temperature. Subsequently, the membranes were washed again with TBST for three times, each time 15 min. The blots were visualized using an enhanced chemiluminescence detection reagent.
Statistical analysis
The identification and verification results were expressed as mean ± standard deviation. SPSS Version 20.0 (SPSS Software, Chicago, IL, USA) was used for statistical analysis, and Student's t-test was used for comparison between groups. P < 0.05 was considered statistically significant.
ResultsBehavioral observations
Rats in the Guasha and model groups showed irritability, biting limbs, repeated licking of the hind claws, and decreased consumption. In the scraping group, the symptoms improved after the later intervention. There were no deaths, spinal cord injury-related symptoms, significant inflammatory response, or scar tissue. In addition, the wound healed well in all rats.
Paw withdrawal thermal latency test results
As presented in [Table 1], PWTL in both groups dropped significantly after the application of NP to the L5 DRG on the day before treatment (P < 0.05).
Isobaric tags for relative and absolute quantitation quantitative of differentially expressed proteins
Compared to the model group, 198 (upregulated: 75, downregulated: 123), 182 (upregulated: 71, downregulated: 111), and 170 proteins (upregulated: 65, downregulated: 105) were identified as differentially expressed proteins in the Guasha group at three respective courses of treatment.
Bioinformatic analysis
As shown in [Figure 1], [Figure 2], [Figure 3], the functional classification of the proteins associated with Guasha was elucidated using GO analysis. After one course of treatment, 10 MFs, 13 CMs, and BPs were implicated. After two courses of treatment, 11 MFs, 13 CMs, and 19 BPs were involved. After three courses of treatment, 10 MFs, 15CMs, and 18 BPs were found. The major subcategories of MF, CM, and BP in the different courses of treatment were identical. The top three MFs were binding (48%, 44%, and 45%), catalytic activity (22%, 21%, and 25%), and MF regulators (5%, 9%, and 6%). For CM, the main subcategories were cell components (28%, 27%, and 27%), organelles (18%, 18%, and 18%), and organelle components (14%, 13%, and 13%). Based on the BP classification results, the top three process proteins involved were cellular processes (19%, 16%, and 20%), biological regulation (15%, 15%, and 16%), and single-organism processes (15%, 14%, and 15%).
Figure 1: GO annotation of identified serum proteins according to molecular functions after one (1), two (2) and three (3) courses of treatment (Guasha group, n=6 per time point) or no treatment (model group, n=6 per time point)
Figure 2: GO annotation of identified serum proteins according to cellular component after one (1), two (2) and three (3) courses of treatment (Guasha group, n=6 per time point) or no treatment (model group, n=6 per time point)
Figure 3: GO annotation of identified serum proteins according to biological processes after one (1), two (2) and three (3) courses of treatment (Guasha group, n=6 per time point) or no treatment (model group, n=6 per time point)In addition, with the use of IPA interaction analysis, compared to the model group, a total of 306, 245, and 245 canonical pathways were annotated in the Guasha group at each course of treatment. The canonical pathway with the highest enrichment in the three respective courses of treatment is shown in [Table 2].
Table 2: Pathways of differentially expressed proteins by Ingenuity Pathways Analysis softwareVerification of the differentially expressed protein by western blotting
Several iTRAQ-identified proteins, including Rac1, Orm1, and Hpx, were confirmed by western blotting [Figure 4]. The results showed that all protein expression levels were significantly higher in the Guasha group than in the model group at all three time points. These results were consistent with the protein expression levels observed using the iTRAQ approach, indicating the reliability of the proteomic analysis.
Figure 4: Effects of Guasha intervention on the expression of Rac1, Orm1, and Hpx determined by western blot after treatment (Guasha group, n=6 per time point) or no treatment (model group, n=6 per time point). Data are mean±SD. *P<0.05, **P<0.01 in Guasha versus model groups (Student's t test) Discussion Our previous studies have provided evidence of the anti-inflammatory, immunomodulatory, and neuroprotective effects of Guasha on LDH,[20],[21],[22],[23] but the deeper molecular mechanism still needs to be explored. In the present study, we first employed quantitative proteomics to identify proteins associated with LDH treatment using Guasha in a rat model.
Three observation time points were set up to capture meaningful proteins in the treatment of LDH using Guasha. Using the iTRAQ technique, we found that 198, 182, and 170 proteins in the Guasha group changed significantly at the three time points compared with the model group, and no common differential proteins were detected. The change in protein expression level was relatively independent at different time points, which suggests that the relationship between the duration of treatment and the curative effect of Guasha may not be a simple linear relationship, and the effect of a long course of treatment is not a simple expansion of the effect of a short course of treatment. Therefore, a more complex mechanism may exist. The GO analysis suggested that Guasha could not only regulate the pathological changes related to LDH but also achieve therapeutic effects by stimulating physiological changes. Therefore, Guasha might control the balance of yin and yang of the body macroscopically. On the other hand, proteomic technology could elucidate the mechanism of the effects of Guasha on LDH from the holistic point of view.
By IPA analysis, we found that focal adhesion kinase (FAK) signaling was the signaling pathway with the highest concentration after one course of Guasha. Lian et al.[28] found that FAK signaling regulates angiogenesis in the dorsal horn of the spinal cord and thus relieves the symptoms of chronic inflammatory pain. In addition, FAK signaling promotes cytoskeleton recombination.[29] In our study, the proteins involved in this pathway were Pik3cg, Rac1, Tln1, Map2k1, and Itga4. The expression of Pik3cg and Rac1 was upregulated, while the expression of Tln1, Map2k1, and Itga4 was downregulated. Rac1 is involved in many cellular processes and the regulation of nerve growth, nerve survival, and neurodegenerative processes.[30] It is also a molecular switch in cytoskeletal regulation. Because apoptosis of chondrocytes is related to the pathological process of intervertebral disc degeneration, the cytoskeleton is important in maintaining the morphology and function of chondrocytes.[31] We speculated that Guasha may regulate FAK signaling through Rac1, thus playing a role in neuroprotection, alleviating inflammation, and maintaining the morphology and function of chondrocytes.
The acute-phase response signaling pathway was the signaling pathway with the highest concentration after two courses of treatment. It is a general response of the body to inflammation and may be caused by tissue injury. Activation of this signaling pathway indicates an increase in proinflammatory cytokines and a change in plasma protein concentration. The proteins involved in this pathway were C4a/C4b, Itih3, Tf, Orm1, Itih4, C9, Stat3, and Nr3c1, of which the expressions of Itih3, Orm1, Itih4, and C9 increased. Among them, Orm1 is a transporter that is mainly involved in the process of acute inflammatory reactions and has anti-inflammatory and immunomodulatory effects.[32] Muñoz et al.[33] found that, after spinal surgery, the expression of several inflammatory response proteins (including Orm1) in the serum of patients was upregulated, which is similar to the results of this study, suggesting that Guasha can play an anti-inflammatory role by promoting Orm1. Therefore, we speculate that Guasha regulates the acute-phase response signaling pathway through Orm1 and exerts anti-inflammatory and immunomodulatory effects.
The LXR/RXR activation pathway was the signaling pathway with the highest concentration after three courses of Guasha intervention. Sevastou et al.[34] suggested that the LXR/RXR activation pathway can promote myelin regeneration in mice with encephalomyelitis. Because the myelin sheath can protect neurons and make nerve impulses transmit quickly to neurons, we speculate that Guasha can promote myelin regeneration by activating the LXR/RXR activation pathway, playing a neuroprotective role, and reducing the pain caused by LDH. Hpx, which is involved in the pathway, is a heme-binding protein that has many functions, such as antioxidation, antiapoptosis, and immunomodulation. Thus, Guasha might also reduce the pain caused by LDH by Hpx, which regulates the LXR/RXR activation pathway and plays a role in neuroprotection, antioxidation, and immunomodulation.
ConclusionUsing the iTRAQ technique, we found that the expression of many proteins was changed in many functions after Guasha intervention, which indicated that there were extensive and complex changes involved. Guasha can not only regulate the pathological changes related to LDH, but also achieve therapeutic effects by stimulating physiological changes. Different proteins may successively activate different pathways to induce the response of the body and play a therapeutic role. The three major pathways are FAK signaling, acute-phase response signaling, and LXR/RXR activation. Proteins, such as Rac1, Orm1, and Hpx, may have an important impact in these processes and could be potential target proteins. Furthermore, the identified proteins and signaling pathways were involved in a wide range of processes, which confirmed that the characteristic of scraping is to produce therapeutic effects through multilink, multiway, and multitarget comprehensive action. This offers a new hypothesis and basis for explaining the mechanism of the effect of Guasha therapy on LDH.
Acknowledgments
Min Yang was responsible for the experimental operations and the writing of the manuscript. Gui-Hua Xu guided the design of the experiments and critically revised the manuscript.
Financial support and sponsorship
This research gained support from the National Science Foundation of China (NSFC; No. 81473791) and The Third Open Subject of Nursing, Nanjing University of Chinese Medicine (No. 2019YSHL028).
Conflicts of interest
There are no conflicts of interest.
References
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