Introduction
Lumbar spinal stenosis (LSS), characterized by the narrowing of the spinal canal at the lumbar vertebral segment, is a prevalent condition in older adults, affecting approximately 200,000 individuals in the United States. The most common cause of this disease is degenerative changes in the intervertebral discs, ligamentum flavum, and facet joints. Symptoms of LSS can vary but they often result from neurovascular mechanisms, nerve root excitation, or mechanical compression of the spinal canal. These mechanisms can occur concurrently, leading to neuropathic pain that can significantly impact daily functioning [1–4].
The treatment for spinal stenosis typically targets symptom management rather than addressing the underlying cause, particularly when symptoms persist despite etiological treatment. This approach can have significant psychological consequences, with approximately 20% of patients with LSS developing depression [2,5]. Preclinical in vitro studies and animal models of stroke have demonstrated the efficacy of MLC601 and MLC901. MLC601 is a precursor formulation to MLC901, and both share three main mechanisms of action that modulate the nervous system: neuroprotection, neuroregeneration, and neurorestoration [6]. Increased levels of MLC901 have been shown to modulate the expression of various factors involved in angiogenesis, including hypoxia-inducible factor 1α, erythropoietin, vascular endothelial growth factor, and angiopoietins1/2. These factors play a crucial role in regulating endothelial cell proliferation and differentiation, regression, and vascular permeability. Consequently, the modulation of these factors by MLC901 contributes to its neuroprotective effects [7,8].
Transforming growth factor (TGF-β) is an anti-inflammatory cytokine that plays a crucial role in responding to cell damage, particularly in the context of nervous system function [9]. TGF-β can reduce glial activity in the spinal cord and reduce inflammation, making it a potent inhibitor of neuropathic pain [10]. The analgesic effects of TGF-β are mediated through gene transcription and neuromodulation pathways in the dorsal root ganglion. Specifically, TGF-β increases the expression of endogenous opioids, reduces glial activation, and suppresses the release of proinflammatory cytokines [11]. TGF-β also modulates plasticity and neurite growth, which are important for nerve regeneration, and reduces axonal demyelination in spinal stenosis [12].
Recent unpublished research by Priyanto (Indonesian dissertation repository; https://repository.unhas.ac.id/id/eprint/38604/) has demonstrated that TGF-β1 can increase serum TGF-β levels and enhance miR30C-5P expression. In the context of peripheral nerve injuries, such as lumbar stenosis, TGF-β1 facilitates the recruitment and function of Schwann cells and macrophages, promoting the clearance of myelin debris. TGF-β1 regulates Schwann cell numbers, protects the basement membrane, promotes migration, and facilitates the formation of Bungner’s bands. Additionally, TGF-β1 polarizes M1 macrophages to the M2 phenotype and activates T regulatory cell (Treg) and T helper cell type 2 (Th2), fostering an environment conducive to nerve cell growth. TGF-β1 also stimulates the secretion of neurotrophic factors such as nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF), which are crucial for nerve cell regeneration [13–15]. TGF-β1 has been identified as a target of microRNA-30c-5p (miR-30c-5p). Studies using rat models of sciatic nerve injury have reported significantly elevated levels of miR-30c-5p in the spinal cord, dorsal root ganglia, cerebrospinal fluid, and plasma. In this context, TGF-β1 expression is downregulated by miR-30c-5p, while using a miR-30c-5p inhibitor leads to an upregulation of TGF-β1 following nerve injury. These findings suggest a regulatory mechanism whereby miR-30c-5p modulates TGF-β1 levels in response to nerve damage [16].
There is a paucity of studies investigating the effect of MLC901 administration on serum levels and immunohistochemical expression of TGF-β1, as well as the degree of axonal demyelination, in animal models using circumferential lumbar stenosis (CLS). Thus, this study aims to provide valuable insights into the potential of MLC901 as a restorative therapy for peripheral nerve injury due to CLS.
Materials and Methods
The study employed an experimental, nonrandomized intervention design with a control group. The study was conducted at the Animal Laboratory of the Faculty of Veterinary Medicine, Hasanuddin University, Makassar from March to June 2024. The study protocol was approved by the Health Research Ethical Committee of the Medical Faculty of Hasanuddin University (approval no., 240/UN4.6.4.5.31/PP36/20024; protocol no., UH24030186).
The study utilized 30 male Sprague-Dawley rats aged 3 months and weighing 200–250 g. To achieve a confidence level of >80% and a significance level of 0.05, a sample size of five rats per group was determined using the Federer formula and World Health Organization Research Guidelines for Evaluating the Safety and Efficacy of Herbal Medicine [17].
The experimental animals underwent a 1-week acclimation to standard maintenance and feeding conditions. The animals were then randomly divided into six groups using simple random sampling. Five groups underwent CLS surgery, while one group served as a nonsurgical control (baseline group). In the baseline group, peripheral blood serum samples were collected to assess TGF-β1 levels without conducting CLS surgery. Subsequently, the animals were euthanized, and cauda equina tissue samples were harvested at the lumbar level 5. On the same day, tissue samples were examined for TGF-β1 immunohistochemical expression and axon demyelination histopathology. CLS surgery was performed in the pretreatment group, and peripheral blood serum samples were collected on the 7th postoperative day for TGF-β1 level analysis. Subsequently, vertebral tissue from the compression area was harvested for analysis of TGF-β1 immunohistochemical expression and axon demyelination histopathology on the same day (without administering MLC901/placebo). In the short-treatment placebo group, CLS surgery was followed by an intraperitoneal placebo injection on the 7th postoperative day. Twenty-four hours later (day 8 post-CLS surgery), serum samples were collected for TGF-β1 analysis. The animals were then euthanized, and vertebral tissue from the compression area was harvested for immunohistochemical evaluation of TGF-β1 and axonal demyelination histopathology on the same day. In the MLC901 short-treatment group, CLS surgery was performed, followed by an intraperitoneal injection of MLC901 on the 7th postoperative day. Twenty-four hours later (day 8 post-CLS surgery), serum samples were collected for TGF-β1 level examination, and the animals were euthanized. Vertebral tissue from the compression area was harvested for analysis of TGF-β1 expression and axonal demyelination. In the long-treatment placebo group, CLS surgery was performed, followed by intraperitoneal placebo administration on the 7th day. The placebo was continued via sonde for 7 days, with the same volume administered daily. On the 14th day postsurgery, serum samples were collected for TGF-β1 level examination, and vertebral tissue was harvested for immunohistochemical analysis of TGF-β1 and histopathological evaluation of axon demyelination on the same day. In the long-treatment MLC901 group, CLS surgery was followed by an intraperitoneal administration of MLC901 on the 7th day, continuing via sonde for 7 days with the same daily volume. On the 14th day after surgery, peripheral blood serum samples were collected for TGF-β1 examination, and vertebral tissue was sampled from the compression area for the analysis of TGF-β1 expression and axonal demyelination histopathology, performed on the same day. In all groups except the baseline, surgical wound care and analgesic treatment were provided only on the first day. Subsequently, the experimental animals received primary care and maintenance for 7 days.
Circumferential lumbar stenosis procedure
Circumferential lumbar stenosis procedures were performed in five groups, excluding the baseline group. The modeling of the CLS procedure in this study mimics developmental spinal stenosis, which was validated by Cheung et al. [18] in 2019. Prior to the procedure, the operating area was cleaned and prepared with asepsis using betadine. Anesthesia was administered using ketamine HCl 60 mg/kg body weight (BW) and Xylazine 10 mg/kg BW intraperitoneally. An incision was made from the level of lumbar 4 (L4) to the sacrum. A laminectomy was then performed at lumbar 5 (L5), which is the second vertebra above the sacrum. A Teflon implant (0.75 mm thick, 3 mm wide, and 10 mm long) was inserted around the dura using a guide and secured with a nylon 5.0 suture. Postoperative wound care included treatment with an antiseptic solution to prevent infection, and prophylactic antibiotics (cefotaxime) were administered via intravenous injection.
MLC901 treatment protocol
MLC 901 is a 9-component oral herbal preparation containing 0.80 g Radix astragali, 0.16 g Radix salvia miltiorrhizae, 0.16 g Radix paeoniae rubra, 0.16 g Rhizoma chuanxiong, 0.16 g Radix angelicae sinesis, 0.16 g Carthamus tinctorius, 0.16 g Prunus persica, 0.16 g Radix polygalae, and 0.16 g Rhizoma acori tatarinowii [19]. MLC901 was diluted in 0.9% NaCl to a concentration of 75 mg/mL and incubated at 37°C for 1 hour. The solution was then filtered through a 0.22-μm filter and administered intraperitoneally as a single dose of MLC901 at a concentration of 0.075 mg/μL, a bolus injection of 500 μL (37.5 mg) 7 days after CSS surgery. Subsequently, oral administration of MLC901 was continued at a dose of 68.4 mg/day until the animal was euthanized.
ELISA for measuring serum TGF-β1 levels
Serum TGF β1 levels were measured using the enzyme-linked immunosorbent assay (ELISA) method (Rat TGFB1/TGF Beta 1 ELISA Kit; LSBio, Newark, CA, USA; Cat no., LS-F23561) according to the standard protocol. The optical density value of each sample was determined using a microplate reader (Biomerieux reader 270; bioMérieux, Marcy-l’Étoile, France) set at a wavelength of 450 nm. The TGF-β1 levels were expressed in pg/mL units.
Histopathological examination of axon demyelination
The lumbar vertebrae were placed in a 4% buffered formalin solution for 12 hours at 40°C. The dural sac was then cut approximately 1 cm proximal and distal to the compression site and separated from the vertebrae. The specimens were washed with phosphate buffer solution and postfixed in 2% osmium tetraoxide (OsO4) for 120 minutes at room temperature. The tissues were then dehydrated in a graded ethanol series (30%–100%) and cleared with chloroform. The tissues were embedded in paraffin blocks, and 8-μm-thick transverse sections of the lumbar nerve fibers at the compression site were cut. The sections were mounted on glass slides, dried, and dewaxed. The slides were then stained using Luxol Fast Blue. Histological analysis was performed on the compression area using 3–5 transverse sections.
Immunohistochemical examination of TGF-β1 tissue expression
Initially, 3-μm-thick sections were cut, dried at 37°C, and placed on a slide warmer at 60°C for 30–60 minutes. The sections then underwent gradual deparaffinization and rehydration using absolute alcohol. The pretreatment procedure was performed according to standard protocols, followed by incubation with TGF-β1 Primary Antibody (Affinity Biosciences, London, UK; Cat no., BF8012) for 30–45 minutes. The tissue was then incubated with UltraTek HRP (ScyTek Laboratories, Logan, UT, USA) as a secondary antibody for 10 minutes. Subsequently, the tissue sample was treated with DAB solution (prepared by mixing 1 mL DAB substrate high contrast + 50 μL/1 drop of DAB Chromogen) for 1–5 minutes. In the final stage, the tissue was washed with running water for 5 minutes, counterstained with hematoxylin for 2–3 minutes, washed with running water for 3 minutes, soaked in bluing reagent for 5–10 seconds, and rewashed with running water for 2 minutes. The tissue samples were then dehydrated and cleared with xylol. TGF-β1 expression was quantified based on the area of brown precipitates using the ImageJ application.
Statistical analysis
Data processing and analyses were conducted using IBM SPSS ver. 22.0 software (IBM Corp., Armonk, NY, USA). All measurement results are presented as mean±standard deviation. Normality testing was conducted to determine the appropriate statistical test. Normally distributed data were analyzed using one-way analysis of variance for multigroup comparisons and the independent t-test for comparisons between two groups. Nonnormally distributed data were analyzed using the Kruskal-Wallis test for multigroup comparisons or the Mann-Whitney test for two-group comparisons.
Results
Comparison of serum TGF-β1 levels
Table 1 presents the differences in serum TGF-β1 levels among the treatment groups. Significant differences (p<0.001) were observed between all groups. The highest serum TGF-β1 levels were found in the baseline group (336.17±8.01 pg/mL), followed by the MLC901 long treatment group (322.78±6.27 pg/mL), the MLC901 short treatment group (285.13±3.59 pg/mL), the pre-treatment group (247.26±4.14 pg/mL), the placebo short treatment group (213.68±6.46 pg/mL), and the placebo long treatment group (193.69±6.95 pg/mL). Notably, the CLS model represented by the pre-treatment group showed significantly lower serum TGF-β1 levels compared to the baseline group. In the placebo groups, serum TGF-β1 levels tended to decrease from baseline values within 1–2 weeks after CLS surgery. The MLC901 groups exhibited an increasing trend in serum TGF-β1 levels, approaching baseline values within 1–2 weeks after CLS surgery. Compared to the placebo group, the MLC901 group consistently showed higher serum TGF-β1 levels in short-term and long-term therapy durations. The results of ELISA analysis are illustrated in the graph provided in Appendix 1.
Comparison of tissue TGF-β1 expression based on immunohistochemical examination
Table 2 shows the differences in tissue TGF-β1 expression among the treatment groups. Significant differences (p=0.013) were observed between all groups. The highest tissue TGF-β1 expression was found in the MLC901 long treatment group (4,377.80±2,416.66), followed by the baseline group (3,804.40±1,567.35), the placebo long treatment group (3,172.00±2,490.46), the MLC901 short treatment group (743.60±278.79), the pretreatment group (397.80±355.56), and the placebo short treatment group (350.40±226.30). Notably, the CLS model represented by the pretreatment group showed significantly lower tissue TGF-β1 expression compared to the baseline group. In the placebo groups, tissue TGF-β1 expression tended to increase toward baseline values within 1–2 weeks after CLS surgery. In the MLC901 groups, tissue TGF-β1 expression showed an increasing trend, approaching baseline value within 1 week after CLS surgery, and even surpassing baseline values within 2 weeks after CLS surgery, although this increase was not statistically significant. Compared to the placebo group, the MLC901 group exhibited higher tissue TGF-β1 expression during short-term therapy, but this difference was not observed during long-term therapy. A representative image of the immunohistochemistry examination is presented in Fig. 1.
Comparison of the degree of tissue axonal demyelination based on histopathological examination
A comparison of the level of axon demyelination is depicted in Fig. 2. The group that received longer MLC901 treatment exhibited inflammatory cell and vacuole values closest to those of the baseline group and also had the highest number of Schwann cells.
Table 3 presents the differences in the number of Schwann cells among the treatment groups. Significant differences (p<0.001) were observed between all groups. The highest number of Schwann cells was found in the MLC901 long treatment group (250.00±126.30), followed by the baseline group (100.40±9.76), the MLC901 short treatment group (99.00±72.22), the placebo short treatment group (62.20±39.22), the placebo long treatment group (57.00±26.38), and the pretreatment group (20.60±10.57). Notably, the CLS model, represented by the pretreatment group, demonstrated significantly fewer Schwann cells compared to the baseline group. The number of Schwann cells in the groups receiving placebo was similar to the baseline group within 1–2 weeks after CLS surgery. In the groups receiving MLC901, the number of Schwann cells showed an increasing trend, approaching baseline values within 1 week after CLS surgery, and even surpassing baseline values within 2 weeks after CLS surgery, although this increase was not statistically significant. Compared to the group receiving placebo, the MLC901 group exhibited a higher number of Schwann cells in both short-term and long-term therapy durations, although the difference did not reach statistical significance.
Table 4 presents the differences in the number of inflammatory cells among the treatment groups. Significant differences (p=0.001) were observed between all groups. The highest number of inflammatory cells was found in the MLC901 short treatment group (193.00±109.12), followed by the placebo long treatment group (185.80±61.39), the pretreatment group (169.00±87.16), the placebo short treatment group (155.80±33.48), the MLC901 long treatment group (52.00±10.95), and the baseline group (21.40±6.62). Notably, the CLS model represented by the pretreatment group showed significantly more inflammatory cells compared to the baseline group. In the groups receiving placebo, the number of inflammatory cells showed an increasing trend, deviating further from the baseline group within 1–2 weeks after CLS surgery. In the groups receiving MLC901, the number of inflammatory cells showed a decreasing trend, approaching baseline values within 1–2 weeks after CLS surgery. Compared to the group receiving the placebo, the MLC901 group exhibited fewer inflammatory cells during long-term therapy but not during short-term therapy.
Table 5 presents the differences in the number of vacuoles among the treatment groups. Significant differences (p=0.015) were observed between all groups. The highest number of vacuoles was found in the pretreatment group (326.60±88.07), followed by the MLC901 short treatment group (224.00±60.60), the placebo long treatment group (192.80±116.91), the placebo short treatment group (186.80±85.27), the MLC901 long treatment group (174.60±177.72), and the baseline group (20.20±12.93). The CLS model represented by the pretreatment group demonstrated significantly more vacuoles compared to the baseline group. In the groups that received placebo, the number of vacuoles showed an increasing trend, deviating further from the value in the baseline group within 1–2 weeks after CLS surgery. In the groups receiving MLC901, the number of vacuoles exhibited a decreasing trend in the number of vacuoles, approaching baseline values within 1–2 weeks after CLS surgery. Compared to the group receiving the placebo, the MLC901 group demonstrated similar vacuoles during both short-term and long-term therapy.
Discussion
Histopathological image analysis revealed the occurrence of axonal demyelination in groups with CLS surgical intervention. This was evidenced by a higher number of Schwann cells and lower numbers of inflammatory cells and vacuoles compared to the group without CLS (baseline). These findings are consistent with the research by Cheung et al. [18], showing that CLS surgical intervention can cause severe and progressive axonal demyelination in experimental animals. The CLS method significantly reduces the levels and immunohistochemical expression of TGF-β1 and induces the occurrence of axonal demyelination. Lumbar stenosis-induced damage is linked to the onset of inflammatory responses. Proinflammatory cytokines play a role in axonal damage, enhancing nociceptor activity, increasing sensitivity, and releasing proinflammatory mediators such as NGF, nitric oxide (NO), interleukin (IL)-1, and IL-6 in response to cellular injury. In chronic constriction injury models of neuropathic pain, the expression of genes associated with immune responses and microglial activation can serve as indicators of neuropathic pain [20,21].
Previous studies have investigated the effect of MLC901 on serum TGF-β1 levels in the context of CLS surgical intervention. However, the effect of MLC901 on the immunohistochemical expression of TGF-β1 has not been investigated. MLC901 is a proprietary blend of nine herbal ingredients that has been used in humans; The neuroprotective and neuroproliferative properties of MLC901 are well-established, as detailed in Table 6 [22–30]. MLC901 has been shown to inhibit the increased expression of mirR30c-5p, which in turn increases TGF-β levels and contributes to the inhibition of axonal demyelination [31–33]. This finding is supported by research conducted by Priyanto et al. [33], who observed a decrease in mirR30c-5p levels and an increase in serum TGF-β levels following 7 days of MLC901 administration in animal models of peripheral nerve disorders. In the present study, serum TGF-β1 levels increased significantly after MLC901 administration for 1 day following CLS surgical intervention. Furthermore, routine administration of MLC901 for 7 days resulted in even higher serum TGF-β1 levels compared to the pretreatment group. In contrast, the placebo groups showed lower serum TGF-β1 levels than the pretreatment group at both time points. Consistent with these findings, tissue expression of TGF-β1 was higher after MLC901 administration for 1 day and 1 week. In the context of peripheral nerve injury, TGF-β1 facilitates the recruitment and function of Schwann cells and macrophages to clean myelin debris by regulating the expression of receptor tyrosine kinase and matrix metallopeptidase [34,35]. Furthermore, TGF-β regulates the number of Schwann cells, protects the basement membrane, increases migration, and promotes the formation of the Bungner strip. TGF-β also exerts immunomodulatory effects by polarizing M1 macrophages toward the M2 phenotype, supporting the activation of Treg and Th2 cells, and creating a microenvironment conducive to cell growth. Additionally, TGF-β activates regeneration-associated gene neurons, leading to the release of neurotrophic factors NGF and GDNF, which in turn enhance the growth capacity of nerve cells [13].
The administration of MLC901 in the group with CLS surgical intervention yielded superior benefits. The number of Schwann cells was significantly higher in the MLC901-treated groups, both after 1 day and 4 days of administration, compared to the placebo groups. Furthermore, the MLC901 long treatment group exhibited a lower number of inflammatory cells compared to the pretreatment group, whereas the placebo long treatment group showed no such reduction. Additionally, the group with CLS surgical intervention that received MLC901 for 7 days demonstrated fewer vacuoles compared to the group receiving the placebo. As the number of vacuoles is a histopathological marker for assessing axon demyelination, this finding suggests that MLC901 treatment may mitigate axonal damage [36].
In general, the present study revealed that MLC901 administration following CLS surgical intervention increases serum levels and immunohistochemical expression of TGF-β, as well as reduces axonal demyelination compared to the placebo group.
Interestingly, this study revealed a discrepancy between serum levels and immunohistochemical expression of TGF-β1 in the group with CLS surgical intervention that received the placebo for 7 days. Specifically, while serum levels of TGF-β1 were lower in this group, immunohistochemical expression of TGF-β1 was paradoxically higher compared to the pretreatment group. This elevated immunohistochemical expression of TGF-β1 may represent a compensatory response to worsening nerve damage, namely on the 14th day after CLS surgical intervention, consistent with the findings of Xue et al. [37]. They demonstrated that severe myelin damage begins to occur on the 8th day and peaks on the 12th day after compression surgery, with nerve fibers infiltrated by a substantial amount of inflammatory tissue by the 14th day [37]. The decrease in the inflammatory response over time is correlated with a reduction in TGF-β1 levels. Notably, this study found a significantly higher concentration of inflammatory cells within the long treatment group compared to the placebo group, contributing to a sustained elevation in TGF-β1 expression as detected by Immunohistochemistry. The elevated TGF-β1 levels detected by immunohistochemistry may be attributed to the preferential accumulation of TGF-β1 within damaged nerve tissue, rather than its circulation in the bloodstream. Immunohistochemistry offers a distinct advantage over serum-based assessments for detecting specific antibodies within cells or tissues. This advantage stems from the fact that external or systemic variables, such as sex, hematological disorders, or other confounding factors can influence serum levels. In contrast, immunohistochemistry provides a more precise modality for analyzing the expression of TGF-β1 [38]. Furthermore, Wulandari et al. [39] reported an increase in TGF-β levels, reaching a zenith on day 21 during the remodeling phase in mice models subjected to injury.
This study has several limitations that warrant consideration. First, the absence of an analysis of the active ingredient of MCL901 that has the greatest effect on reducing axonal demyelination is a notable limitation. Furthermore, the study’s translation relevance is limited by the inherent differences between the lumbar stenosis model used in rat subjects and the human spine, including anatomical, biomechanical, and pathophysiological differences. These differences underscore the need for more precise translational approaches in future research to enhance the applicability of findings to clinical contexts. Moreover, the CLS procedure does not apply to human subjects due to ethical considerations. Another limitation is the inability to conduct serial measurements from the same individual sample. Histopathological examination remains the only method to confirm axonal demyelination, as noninvasive techniques that provide the same level of accuracy have not yet been developed. Due to histopathological sampling, this method requires the sample discontinuation postcollection. We have acknowledged these constraints to clarify the challenges faced in this field of research. In addition, further research is needed to elucidate the interaction between remyelination and demyelination in Sprague-Dawley rats.
Conclusions
This study demonstrates that MLC901 can inhibit axonal demyelination following CLS surgical intervention by increasing TGF-β1 levels. Further research is necessary to fully elucidate the mechanisms underlying the effects of MLC901, particularly its anti-inflammatory and neurodegenerative properties. Further research is also needed to identify the active ingredients responsible for reducing axonal demyelination.
Key Points
MLC901 can significantly increase serum transforming growth factor (TGF)-β1 levels following circumferential lumbar surgery.
MLC901 can significantly increase tissue TGF-β1 expression following circumferential lumbal surgery.
MLC901 can significantly alleviate histopathological signs of axonal demyelination following circumferential lumbal surgery.
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