Bogijetong Decoction and its Selected Formulation Relieve the Regulation of Neuropathic Pain by Modulating Anti-Inflammatory Cytokines
Article information
Abstract
Purpose:
This study was conducted to identify the anti-inflammatory activities of Bogijetong decoction (BJD) and BJD-derived Be decoction (BeD) in an animal model of neuropathic injury in which the sciatic nerve was ligated.
Methods:
BJD, BeD, or an isotonic sodium chloride solution vehicle were administrated orally to Sprague-Dawley rats after sciatic nerve ligation under anesthesia. Other Sprague–Dawley rats underwent sham surgery with no drug treatment administered, serving as the sham control group. The anti-inflammatory activities of BJD and BeD were investigated using immunofluorescence staining and Western blot analysis.
Results:
Iba1 expression markedly decreased following BeD treatment in the sciatic nerve and dorsal root ganglion but not in the spinal cord of the sciatic nerve-ligated animals. BeD treatment reduced tumor necrosis factor-alpha and interleukin-6 levels in the sciatic nerve, dorsal root ganglion, and spinal cord. BJD treatment also reduced Iba1, tumor necrosis factor-alpha, and interleukin-6 levels in the same tissues, although it was less effective than BeD.
Conclusion:
BeD may exhibit anti-inflammatory activity in peripheral nerves and the spinal cord and may contribute to alleviating neuropathic pain.
I. Introduction
Neuropathic pain is a neurological disorder associated with neuronal damage and abnormal activity of nonneuronal cells1,2. Neuropathic pain can occur because of viral infection, nutritional deficiency, diabetes, and nerve damage resulting from chemotherapy during cancer treatment3. A growing body of evidence shows that neuropathic pain-related nerve damage is not only confined to the nerve fiber but also involves surrounding nonneuronal cells, including Schwann cells in the peripheral nerve and dorsal root ganglion (DRG), as well as infiltrated macrophages, microglial cells, and astrocytes in the spinal cord1. Although several therapeutic approaches, such as the use of anticonvulsants, antidepressants, and dietary supplements, have been attempted for the treatment of neuropathic pain4, no definitive cure has been established.
The use of herbal medicines for the treatment of neuropathic pain has been explored; however, their efficacy remains controversial5. Bogijetong decoction (BJD) was developed to alleviate pain associated with diabetes and chemotherapy in patients with cancer6,7. BJD regulates the production of molecular factors involved in axonal regeneration and neuroprotection of sciatic nerves after injury8. BJD may play a role in regulating neuropathic pain in an animal model of streptozotocin-induced diabetes and chronic constriction of the sciatic nerve9,10. However, BJD contains as many as 18 different herbal constituents (Table 1), making it difficult to classify and identify the key components. For the experiment screening the active components, BJD constituents were categorized into four subgroups (Ba-Bd) based on traditional Korean medicinal theory (Table 2). Four herbal constituents—Panax ginseng, Angelica gigas, Paeonia lactiflora, and Crassostrea gigas—were selected from these subgroups for the most effective herbal drug to facilitate neurite outgrowth in cultured DRG neurons, leading to the formulation of a new decoction, Be decoction (BeD)9,10. We demonstrated that BJD and BeD are effective in regulating the molecular factors involved in sciatic nerve ligation (SNL). However, it is not known whether the effects of BJD and BeD on neuropathic pain are confined to the site of SNL or whether they act on ascending neural pathways that transmit pain signals to the brain.
Sources of BJD
Subgroups of BJD
In the present study, BJD and BeD were investigated to facilitate axonal regeneration after the peripheral nerve from damage caused by taxol or nerve crush8. It means that BJD and BeD may paly a role in regulating neuropathic pain. Thus, this study was conducted to identify the pain control mechanism of BJD and BeD in an animal model of neuropathic injury in which the sciatic nerve was ligated.
II. Materials and Methods
1. Animals
Sprague–Dawley rats (male, 200-250 g) were purchased from Samtako (Seoul, Korea) and kept in an animal room for approximately 1 week before the experiments. They were placed in an animal room with a regulated temperature of 22 ℃, 60% humidity, and a 12-h light/12-h dark cycle. The animals were provided with commercial chow (Samyang Co., Korea) and drinking water ad libitum. Animal care and all experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Committee on the Use of Live Animals for Teaching and Research at Daejeon University (Approval number : DJUARB2025-001).
2. Drugs
BJD is composed of 18 herbal constituents, as described in our previous study8, and BeD is a reformulated decoction in which Panax ginseng (4 g), Angelica gigas (7.5 g), Paeonia lactiflora (7.5 g), and Crassostrea gigas (8 g) were chosen from the Ba to Bd subgroups classified for all BJD herbal ingredients based on traditional Korean medicinal theory8. BJD was obtained from Daejeon University Korean Hospital (Daejeon, Korea) where the quality control of herbal drugs was in accordance with the Standard of Korean Pharmacopoeia (ver. 9, Korea Food and Drug Administration, Korea). A mixture of dried BJD or BeD was suspended in distilled water for 2 h, boiled for 3 h, and filtered three times using Whatman filter paper (Grade 1, Whatman Inc., Clifton, NJ, USA). The extract was frozen at -70 ℃ for 4 h and freeze-dried for 24 h. The purified material was stored at -20 ℃ and used for the experiment after dilution with physiological isotonic sodium chloride solution (1 mg of extract residue/mL in 0.9% NaCl solution).
3. Animal surgery
The rats were deeply anesthetized by intraperitoneal injection of a single dose of ketamine hydrochloride (80 mg/kg, Yuhan Yangheng Inc., Seoul, Korea) and xylazine hydrochloride (5 mg/kg, Bayer Korea, Ltd., Ansan, Korea). The sciatic nerve was exposed at the mid-thigh, penetrated with a surgical needle, and ligated tightly with 5-0 nylon or silk suture thread (Ethicon, Somerville, NJ, USA) at two-thirds of the transverse surface area. After surgery, the animals recovered from anesthesia and were returned to the animal room. Sham surgery was performed on control animals by making a skin incision to expose the nerve without ligation. The animals were sutured, allowed to recover from anesthesia, and maintained under the same conditions as those in the SNL group.
4. Experimental animal groups and preparation of nerve tissues
The animals subjected to SNL were divided into three groups: one injected with BJD (SNL+ BJD), one injected with BeD (SNL+BeD), and one injected with isotonic sodium chloride solution vehicle (0.9% NaCl; SNL+SAL). Animals undergoing sham surgery without drug treatment served as the control group (sham). BJD (400 mg/kg), BeD (400 mg/kg), or an equivalent volume of isotonic sodium chloride solution vehicle was orally administered daily for 14 d starting on day 7 after nerve ligation. All animals were euthanized with an overdose of ketamine (150 mg/kg) and subjected to tissue preparation for biochemical and histochemical analyses. The sciatic nerve, approximately 1 cm long and covering the proximal and distal portions of the ligation site, was dissected. To isolate the DRG at lumbar levels 4 and 5, the dorsal skin at the lower lumbar region was removed, and the vertebrae covering the nerve roots were carefully dissected. DRGs at levels 4 and 5 were excised from the spinal cord and sciatic nerve. The spinal cord at the lower thoracic and upper lumbar levels was exposed, and the thoracolumbar spinal cord was isolated. The sciatic nerve, DRG, and spinal cord tissues were used for western blotting and immunofluorescence staining. The tissues were frozen and kept at -80 ℃ until further analysis, unless used immediately.
5. Western blot analysis
Sciatic nerve, DRG, and spinal cord tissues were washed with ice-cold PBS and sonicated in 400 μL of Triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM β-glycerophosphate, pH 7.14, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3 VO4, 1% Triton X-100, 10% glycerol, 5 μg/mL leupeptin, 5 μg/mL aprotinin, 3 μM benzamidine, 0.5 mM DTT, 1 mM PMSF). Protein (20 μg) was loaded onto an SDS-polyacrylamide gel for electrophoresis and transferred to a PVDF membrane. The primary antibodies used were anti-Iba1 (mouse monoclonal, Wako, Japan), anti-TNF-α (rabbit polyclonal, 1:800, Abcam, UK), anti-IL-6 (rabbit polyclonal, 1:800, Abcam, UK), and anti-β-actin (mouse monoclonal, 1:10,000, Sigma, USA). The secondary antibodies used were horseradish peroxidase-conjugated anti-mouse IgG (1:5000, Cell Signaling, USA) and horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Cell Signaling, USA). Quantitative analysis of protein bands in X-ray film images was performed using i-Solution software (Image & Microscope Technology, Eon, South Korea).
6. Immunofluorescence staining
Sections of the longitudinal sciatic nerve, DRG, and horizontal spinal cord (20 μm thick) were cut using a cryostat (Leica, Wetzlar, Germany) and mounted on positively charged slides (Fisher, USA). For immunofluorescence staining, the tissue sections and cultured DRG neurons were fixed with 4% paraformaldehyde and 4% sucrose in PBS at room temperature for 40 min, permeabilized with 0.5% Nonidet P-40 in PBS, and blocked with 2.5% horse serum and 2.5% bovine serum albumin for 4 h at room temperature. Sections were incubated with the following primary antibodies: anti-neurofilament-200 (NF-200) (mouse monoclonal and rabbit polyclonal, both 1:1000, Santa Cruz, USA), anti-Iba1 (mouse monoclonal, 1:400, Wako, Japan), anti-GFAP (rabbit polyclonal, 1:1000, Dako, USA), anti-TNF-α (rabbit polyclonal, 1:400, Abcam, UK), and anti-IL-6 (rabbit polyclonal, 1:400, Abcam, UK). The sections were then incubated with rhodamine-conjugated goat anti-rabbit IgG or fluorescein-conjugated goat anti-mouse IgG (1:400, Molecular Probes, USA) in 2.5% horse serum and 2.5% bovine serum albumin for 1 h at room temperature and cover-slipped with gelatin mount medium. Sections were viewed using a Nikon fluorescence microscope, and images were captured using a Nikon camera. Merged images were produced using the layer-blending mode in Adobe Photoshop.
7. Statistical analysis
Data are presented as mean±standard error of the mean. Differences among experimental groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test (SPSS version 21.0). A statistically significant difference was set at *p<0.05, **p<0.01, and ***p<0.001.
III. Results
1. Iba1 production in the sciatic nerve, DRG, and spinal cord is downregulated by BJD and BeD in SNL animals
To investigate the effects of BJD and BeD on inflammatory responses in the sciatic nerve, DRG, and spinal cord of SNL animals, we performed nerve ligation and examined changes in the production of inflammatory cytokines in these tissues. As shown in Fig. 1A, the 5-0 nylon suture thread was more efficient than the silk suture in inducing a pain response, as measured by paw withdrawal frequency in the von Frey test. Thus, nylon sutures were used for nerve ligation in rats for the remainder of the study. Western blot analysis showed that Iba1 protein was weakly expressed in the sciatic nerve of the sham control group but dramatically increased after SNL (Fig. 1B). Iba1 protein levels were decreased by BJD treatment and further reduced by BeD treatment. In the DRG, Iba1 protein was also increased by SNL, which was then decreased by BJD and further downregulated by BeD (Fig. 1C). Immunofluorescence labeling of DRG tissue showed no discernible Iba1 signals in the sham group. However, Iba1 signals were clearly detected in the DRG of SNL animals (Fig. 1D) and were localized primarily at the periphery of NF-200-stained sensory neurons (bottom panel in Fig. 1D). Iba1 signals in DRG tissue were attenuated by BJD and BeD treatment, which was consistent with the decrease in Iba1 band intensity identified by western blotting (Fig. 1C).
Effects of BJD and BeD on the production of Iba1 in the sciatic nerve and DRG of SNL animals.
(A) Comparison of pain response effects between animal groups with nylon and silk sutures for nerve ligation. After nerve ligation, animals underwent paw withdrawal frequency measurement in the von Frey test. n (number of animals)=3 per group. ***p<0.001 vs. Sham group; ††p<0.01 vs. silk-sutured groups. (B and C) Western blot analysis of Iba1 protein in the sciatic nerve (B) and DRG (C) of animals receiving different treatments. Western blotting for β-actin in (B) and (C) was performed as a protein loading control. The numbers above the western blot images represent the band intensity of Iba1 relative to β-actin. (D) Immunofluorescence staining of Iba1 in the DRG tissues of the following animal groups : sham, SNL+ isotonic sodium chloride solution (SAL), SNL+BJD, and SNL+BeD. Enlarged images of the rectangular areas in the SNL+SAL group are shown in the bottom panel. BJD : Bogijetong decoction, BeD : Be decoction, DRG : dorsal root ganglion, SNL : sciatic nerve ligation.
We further examined the effects of BJD and BeD treatment on the spinal cord after SNL. Iba1 protein levels were very low in the sham group but strongly induced in the spinal cord after SNL (Fig. 2A). However, treatment with BJD or BeD did not alter Iba1 levels. Enhanced Iba1 signals were observed in the dorsal gray matter of SNL animals, which were attenuated after BJD or BeD treatment (Fig. 2B). Iba1 signals, possibly reflecting its expression in microglial cells, were detected around NF-200-labeled neurons in the spinal cord (arrow in the bottom panel of Fig. 2B). Measurement of GFAP protein levels did not show remarkable changes among the experimental groups (Fig. 2C), and immunofluorescence labeling of astrocytes with a GFAP antibody did not show clear changes in astrocyte numbers among the groups. However, aggregated patterns of GFAP-labeled cells were observed in horizontal spinal cord sections of SNL animals (arrows in Fig. 2D).
Regulation of glial cell activation in the spinal cord by BJD and BeD treatments.
(A) Western blot analysis of Iba1 protein in the spinal cord. Western blotting for β-actin was performed as a protein loading control. The numbers above the western blot image represent the band intensity of Iba1 relative to β-actin. (B) Immunofluorescence staining of Iba1 protein in transverse spinal cord sections at the low-thoracic level. The sections were double-immunolabeled with NF-200 to visualize individual neurons. Enlarged images of the rectangular areas in the SNL+SAL group are shown in the bottom panel. NF-200-labeled individual neurons are marked by arrows. DGM : dorsal gray matter. (C) Western blot analysis of GFAP protein in the spinal cord. The numbers above the western blot image represent the band intensity of GFAP relative to β-actin. (D) Immunofluorescence staining of GFAP protein in horizontal spinal cord sections. BJD : Bogijetong decoction, BeD : Be decoction, SNL : sciatic nerve ligation.
2. Downregulation of SNL-induced TNF-α and IL-6 production in the sciatic nerve, DRG, and spinal cord by BJD and BeD
Western blot analysis revealed that TNF-α protein levels were very low in the sciatic nerve of the sham control group but were strongly induced by SNL (Fig. 3A). Subsequent treatment with BJD decreased TNF-α band intensity, while BeD reduced it more efficiently. A similar analysis in the DRG revealed that TNF-α levels were very low in the sham control group but were strongly induced by SNL (Fig. 3A). As observed in the sciatic nerve, treatment with BJD and BeD substantially decreased TNF-α production in the DRG (Fig. 3B). Immunofluorescence staining of TNF-α in the DRG revealed no detectable signals in the sham group but showed strong induction in the SNL group (Fig. 3C). TNF-α signals were primarily colocalized with NF-200-stained neurons. Following treatment with BJD and BeD, TNF-α levels were greatly diminished. Enlarged images of TNF-α signals in the DRG of the SNL group revealed subcellular localization in the perinuclear region of NF-200-labeled sensory neurons, surrounded by Hoechst-stained nonneuronal cells (arrows in Fig. 3D). In the spinal cord, TNF-α signals were weakly detected in the dorsal gray matter of the sham control group, but their intensity increased after SNL (Fig. 4A). TNF-α band intensity was slightly reduced by BJD treatment and completely abolished by BeD. Consistent with the western blot results, TNF-α signals were evenly distributed in the dorsal spinal cord of SNL animals but were weakened by BJD and further decreased by BeD treatment (Fig. 4B).
Regulation of TNF-α production in the sciatic nerve and DRG of SNL animals after BJD and BeD treatment.
(A and B) Western blot analysis of TNF-α in the sciatic nerve (A) and DRG (B) of animal groups receiving different treatments. Western blotting for β-actin was performed as a protein loading control. The numbers above the western blot images represent the band intensity of TNF-α relative to β-actin. (C) Immunofluorescence staining of TNF-α and NF-200 signals in DRG sections from different animal groups, as indicated in the figure. (D) Enlarged views of TNF-α signals colocalized with NF-200-stained DRG sensory neurons. Hoechst 33258 staining was used to visualize individual nuclei. TNF-α : tumor necrosis factor-alpha, BJD : Bogijetong decoction, BeD : Be decoction, SNL : sciatic nerve ligation, DRG : dorsal root ganglion.
Induction of TNF-α protein in the spinal cord by SNL and its downregulation by BJD and BeD.
(A) Western blot analysis of TNF-α in the spinal cord of different animal groups. Western blotting for β-actin was performed as a protein loading control. The numbers above the western blot image represent the band intensity of TNF-α relative to β-actin. (B) Immunofluorescence staining of TNF-α and NF-200 signals in spinal cord sections from different animal groups, as indicated in the figure. DGM : dorsal gray matter. TNF-α : tumor necrosis factor-alpha, BJD : Bogijetong decoction, BeD : Be decoction, SNL : sciatic nerve ligation.
We also investigated the regulatory activity of BJD and BeD on the production of the proinflammatory cytokine IL-6. IL-6 was weakly detected in the sciatic nerve of the sham group and markedly increased after SNL (Fig. 5A). Treatment with BJD greatly decreased the protein levels compared to those in the SNL group. IL-6 protein levels further decreased to undetectable levels after BeD treatment. In the DRG, IL-6 protein levels were clearly elevated by SNL and downregulated by BJD and BeD treatments (Fig. 5B). Immunofluorescence labeling showed that IL-6 signals were barely detected in the DRG tissue of the sham group and were clearly visible in the SNL group (Fig. 5C). IL-6 signals were decreased by BJD and BeD in terms of the number of positive cells and signal intensity of individual cells. Enlarged views showed that some IL-6 signals were found in NF-200-stained neuronal cells, whereas others were not colocalized with NF-200-positive neurons but were seen in the Hoechst-stained nuclei surrounding individual DRG neurons (marked arrows in Fig. 5D). In the spinal cord, a low level of IL-6 was detected in the sham control group, which increased after SNL (Fig. 6A). IL-6 production was almost completely abolished following treatment with BJD or BeD. Immunofluorescence staining showed that the IL-6 protein signal in the dorsal gray matter of the spinal cord did not colocalize with NF-200-stained neurons, suggesting that IL-6 was produced by nonneuronal cells. Again, IL-6 protein expression in the spinal cord was decreased by BJD and BeD treatments (Fig. 6B).
Regulation of IL-6 protein in the sciatic nerve and DRG by BJD and BeD treatments in SNL animals.
(A and B) Western blot analysis of IL-6 protein in the sciatic nerve (A) and DRG (B) of animal groups receiving different treatments. Western blotting for β-actin was performed as a protein loading control, and the numbers above the western blot image represent the band intensity of IL-6 relative to β-actin. (C) Immunofluorescence staining of IL-6 and NF-200 signals in the DRG sections prepared from different groups of animals as indicated in the figure. (D) Enlarged views of IL-6 signals colocalized with NF-200-stained DRG sensory neurons in the SNL+SAL group. Individual nuclei were visualized by Hoechst 33258 staining. IL : interleukin, DRG : dorsal root ganglion, BJD : Bogijetong decoction, BeD : Be decoction, SNL : sciatic nerve ligation.
Effects of BJD and BeD treatments on IL-6 production in the spinal cord of SNL animals.
(A) Western blot analysis of IL-6 protein in the spinal cord of animal groups receiving different treatments. Western blotting for β-actin was performed as a protein loading control. The numbers above the western blot image represent the band intensity of IL-6 relative to β-actin. (B) Immunofluorescence staining of IL-6 and NF-200 signals in the spinal cord sections prepared from different animal groups as indicated in the figure. DGM : dorsal gray matter. BJD : Bogijetong decoction, BeD : Be decoction, SNL : sciatic nerve ligation, IL : interleukin.
IV. Discussion
In the present study, the effects of BeD and BJD on the regulation of inflammatory responses in peripheral nerves and the spinal cord were investigated in an animal model of neuropathic pain. To this end, we administered BJD and BeD for 2 weeks following 1 week of SNL in rats and found that BeD and BJD attenuated the levels of inflammatory cytokines induced by SNL in the DRG, spinal cord, and sciatic nerve. The present data suggest that BeD and BJD may attenuate neuropathic pain by regulating the production of inflammatory cytokines in the afferent fibers of the sciatic nerve.
Treatment with BJD and BeD facilitated axonal regeneration after peripheral nerve injury8, and BJD and BeD also increased BDNF-TrkB signaling in sciatic nerve fibers connected to DRG sensory neurons after nerve ligation10. In the present study, we investigated the neuropathological effects of BJD and BeD on the spinal cord, where synaptic transmission of pain signals occurs. Our data clearly showed that SNL induced the production of the proinflammatory cytokines TNF-α, IL-6, and Iba1 proteins in the DRG and spinal cord, as well as in the sciatic nerve. Iba1, also known as allograft inflammatory factor 1, is a 17-kDa protein that is expressed in macrophages and microglial cells11,12. Given that Iba1 is solely expressed in the microglial cells of the central nervous system and that its production is upregulated by injury signals13, Iba1 is commonly used as a marker of neuroinflammation in the brain14. In the present study, Iba1 expression was strongly induced in the sciatic nerve after SNL, suggesting its presence in infiltrating macrophages. However, the possibility that Iba1 signals originate from microglial cells invading the sciatic nerve cannot be excluded, as microglial cells have been observed in the DRG after peripheral nerve injury14,15. SNL-linked infiltration and activation of macrophages and microglial cells in the sciatic nerve correlate well with increases in the production of TNF-α and IL-6. Our data further showed that TNF-α signals were mostly colocalized with NF-200-positive DRG neurons, whereas microglia and macrophages, possibly activated by SNL, were positive for IL-6 protein signals. Whether reciprocal interactions between DRG neurons and microglial cells contribute to the expression and secretion of specific types of inflammatory cytokines remains unclear.
The present study further demonstrates that Iba1 expression and the production of TNF-α and IL-6 inflammatory cytokines were elevated in the spinal cord of SNL animals. Notably, Iba1 signals were clearly observed in the dorsal gray matter of the spinal cord, where sciatic afferent fibers transmit synaptic inputs to interneurons (for instance, secondary sensory neurons in the substantia gelatinosa conduct pain signals through the spinothalamic tract to the brain). Iba1-labeled microglial cells were in close contact with spinal neurons, implying a chemotactic interaction between neurons and microglial cells via fractalkine binding to its receptor CX3CR1 and purinergic signaling16-19. SNL signals may be linked to the retrograde transport of neurotrophic signals, such as BDNF-TrkB, based on the observation that BDNF levels were upregulated in the ligation area, while TrkB protein levels were increased in the neuronal cell bodies in the DRG10. In response to SNL, the axonal terminals of the central branch of DRG neurons may secrete chemokines that mediate their interaction with adjacent microglial cells. Additionally, chemokine signaling18 in the spinal cord synaptic zone may lead to glial cell activation and increased production of inflammatory cytokines, such as TNF-α and IL-6. Further studies identifying the mediators of inflammatory cells, such as infiltrated macrophages, microglial cells, and astrocytes, will be crucial for understanding the molecular basis of neuropathic pain.
These data show that BJD and BeD are involved in the regulation of SNL-mediated inflammatory responses. Iba1 levels were reduced in the sciatic nerve and DRG of BeD- and BJD-treated animals, with BeD having a particularly strong effect. However, Iba1 levels in the spinal cord, which were increased by SNL, were not altered by BeD or BJD when assessed via western blot analysis. By contrast, immunofluorescence images showed decreased Iba1 signals in the dorsal spinal cord, suggesting regional variation in Iba1 production within the spinal cord. Since Iba1 production is closely related to the activation of microglial cells and macrophages18, the reduction of Iba1 by BeD and BJD may be related to suppressed activation of macrophages and microglial cells in the peripheral nerve and spinal cord. As discussed previously, chemokine signaling may mediate microglia-macrophage interactions with neurons. Further studies on the role of BJD and BeD in the regulation of chemokine signaling pathways will be useful for understanding their mode of action in neuroinflammation. Additionally, it is worth investigating whether BJD and BeD exert differential effects on the activation of M1 and M2 macrophages in relation to neuropathic pain. Macrophages exist in two subtypes: proinflammatory M1 and anti-inflammatory M220,21. M1 macrophages play a major role in acute inflammatory responses, whereas M2 macrophages are more important for anti-inflammatory functions and pain control22,23. Whether the transition from M1 to M2 macrophage activation is influenced by BeD and BJD administration in SNL animals remains to be explored. Collectively, the downregulation of Iba1 production by BeD and BJD may contribute to the regulation of inflammatory responses in the sciatic nerve, DRG, and spinal cord. A hypothetical scheme illustrating the effects of BeD and BJD on the regulation of SNL-induced inflammation in the sciatic nerve, DRG, and spinal cord is shown in Fig. 7.
A hypothetical schematic showing the regulation of the production of inflammatory cytokines in the sciatic nerve, DRG, and spinal cord after the administration of BeD and BJD in SNL animals.
A sequence of inflammatory responses beginning in the sciatic nerve following SNL is depicted as (1)-(3) in red. BJD : Bogijetong decoction, BeD : Be decoction, SNL : sciatic nerve ligation, DRG : dorsal root ganglion.
The present data also show that BeD and BJD effectively attenuate the production of TNF-α and IL-6 proteins. Immunofluorescence labeling of TNF-α and IL-6 in the DRG reveals that the signal intensity of TNF-α is markedly reduced in most individual DRG neurons and in the spinal cord tissue. Overall, it appears that BeD is more effective than BJD in regulating the production of TNF-α and IL-6 in the sciatic nerve, DRG, and spinal cord of SNL animals. Because BeD and BJD are orally administered, the drugs may have reached at least three nerve tissues, possibly with a similar likelihood of penetration. Although the specific components of BeD and BJD that are transported into pathogenic areas to dampen inflammation in the sciatic nerve, DRG, and spinal cord remain unknown, it is speculated that certain critical chemical ingredients may act in a common manner across all three nerve tissues, as the patterns of inflammation regulation in the three areas are similar. However, further studies are essential to verify why BeD is generally more effective than BJD in regulating SNL-induced neuroinflammation.
V. Conclusion
Our study demonstrates that SNL induces inflammation, as evidenced by increased production of Iba1 protein, which is associated with the activation of macrophages and microglial cells, as well as elevated production of the proinflammatory cytokines TNF-α and IL-6 in the sciatic nerve, DRG, and spinal cord. Oral administration of BeD and BJD attenuates the production of these molecules. While these data support the therapeutic potential of BeD, further investigation with broader molecular targets and histological examinations, including analysis of brain areas, is needed to provide more convincing evidence for the use of BeD and BJD.