A standardized extract of Centella asiatica (ECa 233) prevents temporomandibular joint osteoarthritis by modulating the expression of local inflammatory mediators in mice

Abstract Objectives To investigate the effect of a standardized extract of Centella asiatica (ECa 233), which has anti-inflammatory properties, on the local expression of the transient receptor potential vanilloid 1 (TRPV1), the acid-sensing ion channel subunit 3 (ASIC3), and the calcitonin gene-related peptide (CGRP) in the temporomandibular joint (TMJ) structure 21 days after injecting the TMJ with complete Freund’s adjuvant (CFA). Methodology A mouse model was induced by analyzing the CFA-injected TMJ on days 7, 14, and 21. We assessed TMJ histology by the osteoarthritis cartilage grade score. Then, we observed the effect of different ECa 233 concentrations (30, 100, and 300 mg/kg) and of 140 mg/kg ibuprofen doses on TRPV1, ASIC3, and CGRP local expression on day 21. Results Osteoarthritis cartilage scores were 1.17±0.37 and 3.83±0.68 on days 14 and 21, respectively, in the CFA group (n=5). On day 21, TRPV1, ASIC3, and CGRP expression significantly increased in the CFA group. In the ibuprofen-treated group, TRPV1 expression significantly decreased, but ASIC3 and CGRP showed no significant difference. All ECa 233 doses reduced TRPV1 expression, but the 100 mg/kg ECa 233 dose significantly decreased ASIC3 expression. Conclusions TRPV1, ASIC3, and CGRP expression increased in mice with TMJ-OA on day 21. All ECa 233 and ibuprofen doses inhibited pathogenesis by modulating the local expression of TRPV1 and ASIC3. Therefore, ECa 233 was more effective than ibuprofen.


Introduction
Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerative joint disease characterized by varying degrees of inflammation, destruction of the articular cartilage, and resorption of the subchondral bone 1 . It occurs at every age, but especially in people above 40 years of age, and is usually detected in patients who require dental treatment for TMJ-OA pain. 2,3 The structure of the TMJ is composed of synovial tissue, bones, nerves, and blood vessels. Thus, TMJ injuries damage several tissues.
TRPV1 is a polymodal, nonselective cation channel and a member of the transient receptor potential vanilloid family. 4 It is commonly expressed in sensory neurons innervating joints, especially in articular tissues. 5 A previous study reported that the total protein levels of TRPV1 in osteoarthritis (OA) were higher than those found in healthy joints. 6 ASIC3 is a sodium-selective ion channel activated by low extracellular pH and a member of the degenerin sodium channel superfamily. It is a pain modulator expressed in the knee joint, strongly correlated with weight-bearing pain and secondary hyperalgesia. 7 In a hind-paw osteoarthritis model, ASIC3 was expressed in dorsal root ganglia after 21 days. 8 Furthermore, ASIC3 can effectively transduce the prolonged tissue acidosis occurring in osteoarthritis pathology. 9 Synthesis of the calcitonin gene-related peptide (CGRP) is activated by nerve damage and enhanced by inflammatory mediators, such as bradykinin, serotonin, and interleukins (IL)  and IL-22)-released after tissue injury-especially in the cell membrane of chondrocytes. [7][8][9] Moreover, CGRP also promotes TRPV1 upregulation 9,10 and ASIC3 expression. 7,10 TRPV1 and ASIC3 are inflammatory markers triggered by compression injuries, which are the main causes of TMJ-OA. 3,4 CGRP also directly enhances both TRPV1 and ASIC3. In this study, we investigated whether the local expression of these three proteins in TMJ-OA could be used as a marker for target treatment.
Although nonsteroidal anti-inflammatory drugs (NSAIDs) effectively relieve pain and minimize inflammation in TMJ-OA, they may induce peptic ulcers in the gastric epithelium and increase the risk of cardiovascular diseases when used for long periods. 11 Therefore, alternative drugs are necessary and should be investigated.
Our previous study showed that inflammation, which led to TMJ-OA, 21 days after we injected complete Freund's adjuvant (CFA) in the TMJ of mice. Microcomputed tomography showed significant anatomical changes to the condylar head of the joint, indicating bone deformation. 12 Therefore, condylar bone deterioration starts on day 21 and can then be diagnosed as TMJ-OA.
Centella asiatica is a herb that has been used as a medicine for decades in many countries, such as India, China, and even Thailand. C. asiatica has many interesting pharmacological properties, such as rapid wound-healing, antioxidant, and anti-inflammatory properties. Previous studies analyzed C. asiatica as a standardized pure extract called ECa 233 and found it to be minimally toxic. Its active ingredient is triterpenoid glycoside (85%), divided into madecassoside (53%) and asiaticoside (32%); in a ratio of 1.5:0.5. 13 ECa 233 can suppress many inflammatory mediators, including reactive oxygen species, nitric oxide, inducible nitric oxide synthase, cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and IL-1β. 13,14 Bobade,et al. 15   was necessary to evaluate the color value of the picture background. Therefore, the values were measured to randomly select three non-stained areas on the "articular disk," those values were recorded, and used to calculate the mean value. Then, the area whose color value was higher than the background value was selected. Thus, reliable percent values of the proteinpositive stained area were obtained.

Data analysis
All statistical data were analyzed by the Statistical Package for the Social Sciences version 26.0 (IBM, Chicago, IL, USA). Data are expressed as the mean ± standard error. Normal data distribution was examined by the Kolmogorov-Smirnov test. The sham and CFA groups were compared via the independent t-test.
We compared the differences between groups by the one-way analysis of variance (ANOVA) and the Dunnett's test (post hoc test). A p value of less than 0.05 indicated statistical significance.

Results
ECa 233 prevented the development of TMJ-OA Table 1 summarizes the graded score of the rightside TMJ-OA according to the osteoarthritis cartilage histopathology assessment system. The condylar surface of the CFA group gradually deteriorated.
On day 21, the graded score of the CFA group was 3.83±0.69 (n=5). Table 2  ECa 233 reduced ASIC3, CGRP, and TRPV1 expression in the TMJ 21 days after CFA injection On day 21, the CFA group showed increased expression of TRPV1, ASIC3 and CGRP, as observed on the condylar head (independent t-test, n=5, p<0.05).

Discussion
The inflammatory mediators TRPV1, ASIC3, and    can also reduce inflammation, thereby lowering both temperature and acidity. 14 Therefore, administering ECa 233 can reduce the expression of TRPV1 and ASIC3.
Our study also showed that while a concentration expression is not directly related to inflammatory markers such as ASIC3. 2,28 Furthermore, a CFA-induced TMJ-OA mouse model has been proposed. 12 Our study showed that the condylar head of the TMJ changed after CFA induction.
The evolution of TMJ-OA caused the progressive degeneration of the TMJ, represented by a graded score up to day 21 of the experiment. Therefore, our study confirms this model by grading the TMJ diagnosed with TMJ-OA via the osteoarthritis cartilage histopathology assessment system, which is consistent with previous studies. 12,19 Physicians prescribe anti-inflammatory drugs such as ibuprofen and corticosteroids as the standard clinical treatment of TMJ-OA. 21,29 These drugs can prohibit the local inflammatory mediators located at the cartilage of TMJ, thereby preventing progressive