Standardized Centella asiatica (ECa 233) extract decreased pain hypersensitivity development in a male mouse model of chronic inflammatory temporomandibular disorder

Chronic inflammatory temporomandibular disorder (TMD) pain has a high prevalence, and available nonspecific treatments have adverse side effects. ECa 233, a standardized Centella asiatica extract, is highly anti-inflammatory and safe. We investigated its therapeutic effects by injecting complete Freund’s adjuvant (CFA) into right temporomandibular joint of mice and administering either ibuprofen or ECa 233 (30, 100, and 300 mg/kg) for 28 days. Inflammatory and nociceptive markers, bone density, and pain hypersensitivity were examined. CFA decreased ipsilateral bone density, suggesting inflammation localization, which ipsilaterally caused immediate calcitonin gene-related peptide elevation in the trigeminal ganglia (TG) and trigeminal subnucleus caudalis (TNC), followed by late increase of NaV1.7 in TG and of p-CREB and activation of microglia in TNC. Contralaterally, only p-CREB and activated microglia in TNC showed delayed increase. Pain hypersensitivity, which developed early ipsilaterally, but late contralaterally, was reduced by ibuprofen and ECa 233 (30 or 100 mg/kg). However, ibuprofen and only 100-mg/kg ECa 233 effectively mitigated marker elevation. This suggests 30-mg/kg ECa 233 was antinociceptive, whereas 100-mg/kg ECa 233 was both anti-inflammatory and antinociceptive. ECa 233 may be alternatively and safely used for treating chronic inflammatory TMD pain, showing an inverted U-shaped dose–response relationship with maximal effect at 100 mg/kg.


Results
Descriptive results. Weight. After 28 days of CFA-induced TMD pain, there was no difference in weight between the Sham, Ibu, CFA, 30ECa, 100ECa, and 300ECa groups (Fig. 1). Figure 2 illustrates the time course of behavioral pain responses of the different groups tested by 0.04 g von Frey filament for mechanical hyperalgesia and 0.05 ml cold acetone for cold allodynia. We tested each side 12 times and then recorded the response scores according to the following criteria: no response = 0, occurrence of head withdrawal = 0.25, occurrence of face grooming once = 1, and occurrence of face grooming > 3 times = 1.5. We added all response scores on each side to obtain the total response score; the maximum response score was 18. The Sham group, which was injected with NSS into the right TMJ, showed the lowest pain response scores; the response remained constant at the baseline level throughout the whole experiment. However, the CFA group developed a significant pain hypersensitivity on the ipsilateral side of the CFA injection at post-CFA-injection days 14, 21, and 28 with both von-Frey and cold acetone tests, and on the www.nature.com/scientificreports/ contralateral side at post-CFA-injection day 28 also with both von-Frey and cold acetone tests when compared with the Sham group. In the Ibu, 30ECa, and 100ECa groups, pain hypersensitivity significantly decreased on the ipsilateral side, but not in the 300ECa group, at post-CFA-injection days 14, 21, and 28 with both von-Frey and cold acetone tests. On the contralateral side, pain hypersensitivity significantly decreased in the Ibu, 30ECa, and 100ECa groups at post-CFA-injection day 28 for both von-Frey and cold acetone tests, compared with that in the CFA group. Thus, CFA injection induced pain hypersensitivity, including mechanical hyperalgesia and cold allodynia, initially on the ipsilateral side of the injection and later on the contralateral side. In addition, ibuprofen, which was used as a positive control treatment, and ECa 233 at 30 and 100 mg/kg doses significantly and equally reduced pain hypersensitivity that developed bilaterally, while there was no statistical difference among the three groups at any days measured.

Orofacial pain sensitivity.
Structural changes in TMJ. Figure 3 depicts the bone density of the condylar head of TMJ on the ipsilateral and contralateral sides measured by micro-CT. Although CFA-induced pain hypersensitivity developed early on the ipsilateral side and later on the contralateral side, the bone density of the CFA group significantly reduced only on the ipsilateral side at post-CFA-injection days 21 and 28, compared with that of the Sham group.
Considering that bone density reduction is a major sign of TMJ tissue inflammation, CFA-induced inflamma-  www.nature.com/scientificreports/ tion could cause pathology in the condylar head of the right TMJ, consistent with pain hypersensitivity on the ipsilateral side. Meanwhile, the pain hypersensitivity on the contralateral side seemed not to be induced by the pathology in the contralateral TMJ because no bone density reduction was observed. Compared with the Sham group, only the Ibu and 100ECa groups did not exhibit bone density reduction at post-CFA-injection days 21 and 28. Pain hypersensitivity was also reduced in the 30ECa group; however, this group as well as the CFA and 300ECa groups showed bone density reduction without significant difference among the three groups. Thus, both 30 and 300 mg/kg doses of ECa 233 could not prevent the pathology caused by CFA, whereas 0.14 g/kg ibuprofen and 100 mg/kg ECa 233 effectively prevented the pathology at all of the time points after CFA injection and also reduced pain hypersensitivity.
Inflammation and nociceptive activity changes in TG. Expression of CGRP in TG. Figures 4 and 9 illustrate the expression of CGRP in bilateral TGs. CFA injection immediately and significantly enhanced the CGRP expression in the ipsilateral TG at post-CFA-injection days 3, 7, and 14, but not in the contralateral TG, compared with Sham. These data supported the idea that CFA-induced inflammation on the ipsilateral side immediately activates nociceptors, possibly causing pain hypersensitivity on the same side. Although the 30 and 300 mg/kg doses of ECa 233 could not reduce the enhanced expression of CGRP, ibuprofen and 100 mg/kg ECa 233 effectively prevented the enhanced CGRP expression at post-CFA-injection days 3, 7, and 14 compared with the CFA injection. Thus, 100 mg/kg dose of ECa 233 effectively inhibited inflammation in TG.
Expression of NaV1.7 in TG. Figures 5 and 9 show the NaV1.7 expression in bilateral TGs. In the CFA group, NaV1.7 expression was late yet significantly enhanced in the ipsilateral TG at post-CFA-injection days 21 and 28, but not in the contralateral TG. The immediate enhancement of CGRP and later enhancement of NaV1.7 corresponded well with the early development of pain hypersensitivity on the ipsilateral side. However, the late development of pain hypersensitivity on the contralateral side seemed to be not associated with the contralateral

Discussion
This is the first in vivo study of the standardized extract of C. asiatica (ECa 233) reporting that ECa 233 could attenuate pain hypersensitivity in chronic inflammatory TMD in male mice induced by CFA injection. Despite inducing many side effects, such as gastrointestinal irritation and kidney injury, the NSAID ibuprofen is currently the standard treatment for TMD pain 15 .
After receiving ibuprofen and ECa 233 (30 and 100 mg/kg), the mice had lower pain response scores. However, only ibuprofen and 100 mg/kg ECa 233 could protect the TMJ from bone density changes and prevent the increased expression of inflammation-and nociception-associated proteins in the TG (PNS relay station) and TNC (CNS relay station) of the trigeminal pain pathway. Although the pathophysiology of chronic inflammatory TMD pain remains unclear, other studies hypothesized that TMD results from the local release of inflammatory irritants caused by tissue damage or external irritants such as CFA. Clinically, however, many patients with TMD have no apparent signs of local inflammation or tissue damage 16 , and TMD pain can widely spread within the orofacial area 17 .
In our TMD model, CFA caused an excessive uncontrolled inflammation mimicking the osteoarthritisinduced TMD pain 18 . Owing to the inflammation at the injected TMJ, the TMJ's condylar head degenerated with lowered bone density, leading to osteoarthritis-induced pain hypersensitivity 19 . Consistent with the previous studies, we showed that the unilateral CFA injection induced chronic inflammation in the ipsilateral joint and nervous system (both PNS and CNS); hence, hyperalgesia and allodynia occur on the ipsilateral side. However, www.nature.com/scientificreports/ through our lengthened observation into the chronic phase of pain (28 days after the CFA injection), our study also demonstrated changes that occurred in the contralateral CNS, causing pain hypersensitivity on the contralateral side, as first reported by our group recently, indicative of a mirror-image pain 20 . When stimulated by noxious stimuli, CGRP drives the sensitization at TG and TNC in the chronic phase of the TMD pain, as evidenced by the increased expression of NaV1.7 in TG and that of p-CREB in TNC. In a previous study, injecting CGRP into the rat's TMJ promoted changes in p38 MAP kinases and ERK in TG and c-Fos in TNC after 2 h of injection, indicating peripheral and central sensitizations, respectively 21 . Meanwhile, pain hypersensitivity and neuronal activity in the trigeminal system in response to CFA-induced pain in the masseter muscle were attenuated by a CGRP antagonist 22 . In our study, CGRP was enhanced only in the early stage of inflammation in the ipsilateral TG and TNC, but not in the contralateral TG and TNC; thus, the pain signals stimulating the contralateral TNC (as evidenced by the late enhancement of the expressions of p-CREB and activated microglia at post-CFA-injection day 28) would originate from the ipsilateral TNC or other higher relay stations, eventually producing pain hypersensitization at post-CFA-injection day 28 on the contralateral side. Therefore, our data supported that TMD pain could occur despite the lack of inflammatory signs as in the mirror-image pain. www.nature.com/scientificreports/ In particular, NaV had implicated chronic pain including NaV1.7, NaV1.8, NaV1.9. However, in terms of insight into function, NaV1.7, which is related to communication in nociceptive peripheral afferents, has a hereditary loss-of-function mutation that triggers pain hypersensitivity without causing other neurodevelopmental changes 23 . In an animal model, successful NaV1.7 suppression in the lumbar dorsal root ganglia was associated with decreased thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no modifications to the animals' regular motor function 23,24 . Therefore, in our study the increased expression of NaV1.7, a pain hypersensitivity marker in TG, would lower stimulation threshold and provoke ectopic discharge, causing peripheral sensitization 3 . Moreover, activation of p-CREB, a molecular marker of pain transmission and pain hypersensitivity in TNC 4 , may be a key step for developing a synaptic plasticity-dependent activity that ultimately increases pain perception 25 . The role of activated microglia in TNC and spinal cord is related to pain hypersensitivity, particularly mechanical hyperalgesia following inflammation 26 . Persistent PNS inflammation is a major cause of activated microglia in CNS. Activated microglia release proinflammatory cytokines and chemokines, leading to widespread inflammatory responses. Our study revealed expressions of NaV1.7, p-CREB, and activated microglia in the 100ECa and Ibu groups significantly decreased compared with that in the CFA group. Thus, the 100-mg/kg dose of ECa 233 could prevent pain sensitization, possibly by reducing the CGRP and NaV1.7 expressions, thereby reducing the behavioral pain responses and pain hypersensitivity. www.nature.com/scientificreports/ Consistent with other supporting results, C. asiatica extracts and active ingredients had shown both antiinflammatory and antinociceptive effects. For example, 100 mg/kg dose of C. asiatica inhibited foot edema in carrageenan and prostaglandin-induced hind foot pain in rats, similar to the activity of ibuprofen treatment 8 . C. asiatica also significantly decreased proinflammatory cytokine (TNF-α and interleukin 6) expression in lipopolysaccharide-induced HEK-293 cell inflammation 27 . Moreover, oral madecassoside administration could reduce the clinical pain scores of mice with an induced rheumatoid arthritis and also minimize prostaglandin, COX-2, and proinflammatory cytokine infiltrations 27 . Indeed, madecassoside promotes anti-inflammation and protects the joint from destruction. Moreover, asiaticoside has an antinociceptive effect, seen when oral treatment of asiaticoside reduced pain in mice with capsaicin-induced paw pain 28 . Therefore, ECa 233, which contains ≥ 80% w/w of triterpenoid glycosides with a 1.5:1 ratio of madecassoside and asiaticoside, possesses both anti-inflammatory and antinociceptive activities, resulting in the reduction of inflammatory responses in both PNS and CNS and behavioral pain responses; however, the effective dose of both activities demonstrated an inverted U-shaped dose-response relationship with the maximal effect at 100 mg/kg dose.
At a low dose of 30 mg/kg, ECa 233 showed an effect comparable with that of 100 mg/kg ECa 233 on pain hypersensitivity alleviation, but it did not reduce the expression of inflammation-associated proteins; thus, the antinociceptive activity of ECa 233 started to manifest at the low dose. The 30 mg/kg dose of ECa 233 contains approximately 15 mg/kg dose of madecassoside and 10 mg/kg dose of asiaticoside 9 . Madecassoside at 40 mg/kg (not 10 and 20 mg/kg) could reduce inflammation, thereby lowering pain scores and protecting the joint from destruction; hence, madecassoside exhibits an anti-inflammatory activity 27 . Meanwhile, asiaticoside at 10 mg/kg (not 5 mg/kg) could alleviate pain in capsaicin-induced pain; hence, asiaticoside has an antinociceptive activity 13 . Therefore, the possible mechanism is that at the dose of 30 mg/kg, ECa 233 could exert the antinociceptive effect, thereby reducing the behavioral pain responses; however, it could not reduce inflammatory responses and prevent osteoarthritis development at the CFA-injected TMJ. ECa 233 at the dose of 100 mg/kg containing approximately 49.5 mg/kg concentration of madecassoside and 33 mg/kg concentration of asiaticoside not only reduced the pain responses but also reduced inflammatory responses and prevented osteoarthritis development.
At the low dose of ECa 233 (30 mg/kg), the antinociceptive activity, which might originate from the asiaticoside activity, was perhaps caused by the increased activity of gamma aminobutyric acid (GABA), which is the major inhibitory neurotransmitter of CNS. The activity of glutamic acid decarboxylase, which is involved in GABA synthesis, is increased by an aqueous extract of C. Asiatica 29 . Moreover, asiatic acid, which is an active metabolite of asiaticoside, interacts with GABAB receptor to inhibit synaptic transmission. The increased GABA activity of CNS might cause pain hypersensitivity reduction. However, the GABA expression does not directly   30 and also may not be associated with the expression of inflammationassociated proteins in the pain pathway. Interestingly, high-dose ECa 233 treatment (300 mg/kg) did not exhibit beneficial effects. One of the reasons could be that the plant products contain other constituents, which at a high dose, exert some antagonistic or inhibitory effects. ECa 233 could enhance brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate receptor (NMDAR) in the hippocampus, cerebral cortex, and spinal cord 9 . BDNF facilitated pain transmission and contributed to pain hypersensitivity development 31 via the postsynaptic NMDAR to modulate nociceptive signaling in TNC 32 . After BDNF antagonist injection in mice with formalin-induced pain, pain hypersensitivity decreased 33 . Intrathecal NMDAR antagonist injection in nerve injury-induced neuropathic pain reduced pain hypersensitivity 34 . Therefore, BDNF and NMDAR were involved in some aspects of pain hypersensitivity, possibly in the central sensitization. Thus, at a high dose (300 mg/kg), ECa 233 may facilitate pain transmission, counteracting its antinociceptive and anti-inflammatory effects. All these mechanisms could perhaps inhibit the beneficial effects of the major constituents, possibly owing to which the highest dose of ECa 233 exhibited results similar to those of the control group.
Therefore, the inverted U-shaped dose-response relationship of ECa 233 might indicate one of the factors responsible for pain hypersensitivity reduction. Hence, the effective dose of C. asiatica extract should be limited to moderate doses to achieve beneficial effects in chronic inflammatory TMD pain. The recommended dose of ECa 233 in this study is 100 mg/kg, which showed both antinociceptive and anti-inflammatory effects by preventing the anatomical changes in the condylar head of TMJ and hindering the expression of inflammation-and nociception-associated proteins in the TG and TNC. These findings provide basic knowledge of ECa 233 effects on inflammation and nociception, supporting ECa 233 development as an antinociceptive and anti-inflammatory drug or a dietary supplement for pain treatment.
Our study's limitations include not using the rotarod test for evaluating ECa 233 additional behavioral side effects, and consideration of the subjective meaning of pain. Moreover, NaV implicated in pain such as NaV1.8, NaV1.9 in the trigeminal pathway should be further studied.
In conclusion, C. asiatica has antinociceptive and anti-inflammatory effects on chronic inflammatory TMD pain within a specific therapeutic range. Despite the very limited therapeutic dose of ECa 233, it has potent effects and a high safety profile. Therefore, ECa 233 can be safely used as an antinociceptive and anti-inflammatory agent in chronic pain management, although further investigation is still required.

Methods
Animals. We used 6-week-old healthy male mice (n = 144, weighing 25-30 g, which were purchased from the Nomura Laboratory Animal Center, Thailand. All mice were housed under conditions of controlled humidity (40%-70%), temperature (23 °C ± 1 °C), and light (12 h day/12 h night cycle), with free water and food access. All study protocols were performed in accordance with relevant guidelines and regulations and were approved by the Animal Care and Use Committee of the Faculty of Medicine Siriraj Hospital, Mahidol University (Approval No.: SI-ACUP014/2561). All experimental procedures conformed to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for animal experiment 35 . The sample size was estimated to provide 80% power (1 − β) with a 95% confidence interval (α = 0.05) 36 . According to previous study, CFA-induced TMJ pain animal model was treated with ibuprofen treatment as a positive control 13,37 . A total of 144 mice were used for the study, randomly divided into six groups (n = 24/group): www.nature.com/scientificreports/ Behavioral test. We performed two series of behavioral pain response tests [using 0.04 g of von Frey filament for mechanical hyperalgesia and 0.05 ml of cold acetone (− 5 °C) for cold allodynia with an interval of at least 30 min. At pre-CFA-injection day and post-CFA-injection days 3, 7, 14, 21, and 28, the mice underwent tests at their whisker pads of both ipsilateral and contralateral sides. We tested each side 12 times and then recorded the response scores according to the following criteria: no response = 0, occurrence of head withdrawal = 0.25, occurrence of face grooming once = 1, and occurrence of face grooming of > 3 times = 1.5 18,20 . We added all response scores on each side to obtain the total response score; the maximum response score was 18. Inflammatory and nociceptive activities in TG and TNC. The mice were deeply anesthetized and perfused with the same conditions used for the micro-CT. We located TG and TNC using the brain atlas of Paxinos and Keith 40 . TG and TNC were then extracted bilaterally and immersed in 4% paraformaldehyde in PBS (0.1 M, pH 7.4) immediately. Tissue processing and paraffin embedding procedures were also performed. We transversely sliced 5-μm thick sections using a microtome (Global Medical Instrumentation Inc., Ramsey, MN, USA) and deparaffinized them before immunostaining by immunohistochemistry. The TG paraffin sections were stained with neuroinflammatory markers rabbit monoclonal anti-NaV1.7 antibody (AB5390; Merck KGaA, Darmstadt, Germany) and rabbit polyclonal anti-CGRP antibody (SC-57053; Santa Cruz Biotech, Dallas, TX, USA), whereas the TNC sections were immunolabeled with a rabbit polyclonal anti-p-CREB antibody (SC-81486; Santa Cruz Biotech, Dallas, TX, USA), rabbit polyclonal anti-CGRP antibody (SC-57053; Santa Cruz Biotech, Dallas, TX, USA), and rabbit polyclonal anti-OX42 antibody (SC-52; Santa Cruz Biotech, Dallas, TX, USA) for the expression of activated microglia. Briefly, the TG and TNC sections were warmed with antigen retrieval solution (citrate buffer; Dako, Glostrup, Denmark) in a microwave oven. The endogenous peroxidase activity was inhibited with Dako Peroxidase Blocking Reagent (Dako) for 5 min and then blocked with an antibody diluent (Dako) for 10 min to prevent nonspecific staining. Further, we incubated the sections with the primary antibodies at 4 °C overnight, followed by the secondary antibody (goat anti-rabbit-IgG antibody conjugated with horseradish peroxidase). We used the EnVision™ Detection System (Dako) for signal visualization and a light microscope (Carl Zeiss Microimaging GmbH, AxioVision 40 version 4.8.2.0, Germany) for peroxidase activity site detection. We also evaluated the nociceptive activity in TG and TNC individually and independently and blindly analyzed each datum by the mean number of positive neuron expression at each time point.

Micro
For TG analysis, we used three mice from each group at each time point and selected five sections per TG tissue (5th, 10th, 15th, 20th, and 25th sections) of each mouse. We counted the neurons in each whole TG section and then summed and averaged the numbers of counted neurons from all sections in one mouse. The ratio of positive neurons per total number of neurons and the mean value of each experimental group (n = 3/group/time point) were calculated. For TNC analysis, we used the same three mice from each group at every time point and selected five sections per TNC tissue (5th, 10th, 15th, 20th, and 25th sections) of each mouse. We counted the positive cells (brown dots) at laminas I and II of the TNC sections and then summed and averaged the numbers of brown dots from all sections in one mouse. Thereafter, the mean value of each experimental group (n = 3/ group/time point) was analyzed.
Statistical analysis. All statistical data were analyzed using SPSS version 26.0 (IBM, Chicago, IL, USA).
The Kolmogorov-Smirnov test was used for testing normal data distribution. Possible statistically significant differences between groups were determined using two-way analysis of variance with Fisher's least significant difference post hoc test. All analyses were performed using the two-tailed hypothesis testing method. In addition, P < 0.05 was considered statistically significant. Data are shown as mean ± standard error of the mean.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on a reasonable request.