WGA-M001, a Mixture of Total Extracts of Tagetes erecta and Ocimum basilicum, Synergistically Alleviates Cartilage Destruction by Inhibiting ERK and NF-κB Signaling

Tagetes erecta and Ocimum basilicum are medicinal plants that exhibit anti-inflammatory effects against various diseases. However, their individual and combined effects on osteoarthritis (OA) are unknown. Herein, we aimed to demonstrate the effects of T. erecta, O. basilicum, and their mixture, WGA-M001, on OA pathogenesis. The administration of total extracts of T. erecta and O. basilicum reduced cartilage degradation and inflammation without causing cytotoxicity. Although WGA-M001 contained lower concentrations of the individual extracts, it strongly inhibited the expression of pathogenic factors. In vivo OA studies also supported that WGA-M001 had protective effects against cartilage destruction at lower doses than those of T. erecta and O. basilicum. Moreover, its effects were stronger than those observed using Boswellia and Perna canaliculus. WGA-M001 effectively inhibited the interleukin (IL)-1β-induced nuclear factor kappa-light-chain-enhancer of the activated B cell (NF-κB) pathway and ERK phosphorylation. Furthermore, RNA-sequence analysis also showed that WGA-M001 decreased the expression of genes related to the IL-1β-induced NF-κB and ERK signaling pathways. Therefore, WGA-M001 is more effective than the single total extracts of T. erecta and O. basilicum in attenuating OA progression by regulating ERK and NF-κB signaling. Our results open new possibilities for WGA-M001 as a potential therapeutic agent for OA treatment.


Introduction
Degenerative joint diseases, also known as osteoarthritis (OA), are significant and have become an increasingly prevalent health concern in the aging world population [1].OA is a progressive condition characterized by gradual deterioration of the protective cartilage present within joints, resulting in damage to the underlying bones and ligaments [2].The degeneration causes inflammation and pain, making it a critical disease to manage as the number of affected patients continue to increase [3].Cartilage is a specialized connective tissue that plays a crucial role in supporting and cushioning joints, and provides a smooth surface for frictionless movement [4].It is composed of chondrocytes, extracellular matrix, and molecules produced by the chondrocytes, such as collagen, proteoglycans, and glycosaminoglycans [5,6].In OA, interleukin (IL)-1β binds to specific receptors on the surface of chondrocytes, activating intracellular signaling pathways [7], which lead to the upregulation of matrix metalloproteinases (MMPs) and aggrecanases-enzymes responsible for breaking down the extracellular matrix of cartilage, including collagen and proteoglycans [8][9][10].Moreover, the upregulation of COX-2 and IL-6 enhances inflammatory responses [11,12], resulting in an imbalance between matrix degradation and synthesis, which exacerbates cartilage erosion [13,14].
In addition to surgery, drug therapy is commonly used to treat OA [15], which primarily includes medications with analgesic and anti-inflammatory properties [15].However, a definitive cure for OA has not been developed [16].Non-steroidal anti-inflammatory drugs are the most widely used class of drugs for OA treatment [17].However, these drugs have several potential side effects, which make their long-term use challenging [18].Thus, there is considerable interest in using natural products with chondroprotective and anti-inflammatory activities as functional foods that can protect the cartilage from deterioration [19].
The development and progression of OA occur via two important signaling pathways: the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chainenhancer of activated B cell (NF-κB) signaling pathways [20].The MAPK signaling pathway involves a serine/threonine protein kinase that is widely present in eukaryotic cells and can be activated by IL-1β [21].Upon activation, MAPK transmits extracellular signals to the nucleus to regulate the activity of several transcription factors, resulting in the altered expression of genes encoding MMPs, COX-2, and IL-6 [22][23][24].NF-κB is a well-known inflammatory signaling pathway that is also activated by IL-1β [25,26].Activation of this pathway causes phosphorylation of p65 and IκBα, which results in the translocation of NF-κB into the nucleus, where it subsequently binds to specific sequences within the promoter regions of the target genes encoding pro-inflammatory mediators like COX-2, MMPs, and IL-6 [27].Both the MAPK and NF-κB signaling pathways contribute to disease pathogenesis in OA by promoting inflammation, cartilage breakdown, and other pathological changes within joints [21,26].Therefore, modulation of MAPK and NF-κB signaling may prove beneficial in the treatment of OA.
Tagetes erecta, commonly known as marigold, has been used for medicinal purposes since pre-Hispanic times [28].It is used to treat digestive disorders such as abdominal pain, diarrhea, vomiting, and indigestion, as well as to treat inflammatory conditions such as bronchitis [29].Ocimum basilicum, also known as basil, has diverse applications in traditional medicines, pharmaceuticals, cosmetics, and as a functional food [30].It contains phenolic and flavonoid compounds that exhibit several pharmacological effects, such as antioxidant, anti-inflammatory, and analgesic effects, making it effective against chronic inflammation and immune-related diseases [31].Although both T. erecta and O. basilicum are medicinal plants commonly used as dietary supplements [30,32], their function in OA is largely unknown.Herein, we demonstrate that WGA-M001, a mixture of T. erecta and O. basilicum extracts, strongly attenuates OA development through the inhibition of gene signatures related to ERK and NF-kB pathway activation, compared to the attenuation provided by a single extract of T. erecta or O. basilicum.Moreover, the effect of WGA-M001 strongly suggests the possibility of OA treatment in vitro and in vivo.

Therapeutic Effects of T. erecta and O. basilicum under In Vitro Conditions Mimicking OA
T. erecta and O. basilicum were tested on mouse articular chondrocytes for cytotoxicity analysis.Mouse articular chondrocytes were treated with various concentrations of T. erecta or O. basilicum extract for 24 h.Through lactate dehydrogenase (LDH) assay, it was found that concentrations up to 200 µg/mL did not affect chondrocyte viability (Figure S1A,B).Thus, use of up to 200 µg/mL of T. erecta and O. basilicum extract was suggested to be safe on chondrocytes.The inhibitory effect of T. erecta and O. basilicum on pathogenic factor expression in IL-1β-treated cells in vitro was further analyzed.T. erecta inhibited the transcription (Figure 1A,B) and protein levels (Figure 1C-E) of MMP3, MMP13, COX-2, and IL-6 in a dose-dependent manner.Moreover, compared to T. erecta, O. basilicum had a similar effect on the IL-1 β treated chondrocytes (Figure 1F-I).Although T. erecta and O. basilicum showed similar effects in inhibiting catabolic factor expression, treatment with O. basilicum had a stronger inhibitory effect on pathogenic factor expression than that associated with treatment with T. erecta.Thus, the data suggested that an appropriate proportion of the mixture can strongly inhibit OA pathogenesis by suppressing the expression of catabolic factors.The therapeutic effects of WGA-M001 were then examined using a mixture with an appropriate ratio of T. erecta to O. basilicum, under in vitro conditions that mimic OA.We also demonstrated that WGA-M001 had no cytotoxic effects on mouse articular chondrocytes as observed with respect to T. erecta and O. basilicum (Figure S1C).

WGA-M001 Effectively Attenuates Catabolic Factor Expression
WGA-M001 (100 µg/mL and 200 µg/mL) was used with IL-1β as co-treatment of mouse chondrocytes.The expression of catabolic factors was examined using biochemical analyses.We demonstrated that the transcription levels of MMP3, MMP13, COX-2, and IL-6 were suppressed in IL-1β-treated conditions that mimic OA (Figure 2A,B).The expression level of MMP3, MMP13, COX-2, and IL-6 increased with the IL-1β treatment, compared to the levels in the control, whereas IL-1β induced catabolic factors decreased in the presence of WGA-M001 in a dose-dependent manner.Moreover, the decrease in the level of catabolic factor caused by WGA-M001 (200 µg/mL) was greater than the decrease associated with the same concentration of single extract T. erecta or O. basilicum in chondrocytes.This data collectively suggested that WGA-M001 has an inhibitory effect on catabolic factor expression, compared to T. erecta and O. basilicum treatment.The similarity between the in vitro OA mimic phenotypes and in vivo OA mouse experiments was also studied.

T. erecta and O. basilicum Mixtures Show a Synergistical Effect in the DMM-Induced OA Mouse Model
To verify the in vivo functions of WGA-M001, T. erecta, and O. basilicum, we destabilized the medial meniscus (DMM)-induced OA according to the schedule shown in Figure S2.The DMM-induced OA mouse model is a highly representative in vivo model for OA [33,34].WGA-M001, T. erecta, or O. basilicum was orally administered to mice with DMM-induced OA.To determine the severity of OA development, the cartilage destruction using subchondral bone plate (SBP) thickness and osteophyte maturity was examined [35].SBP plays an important role in articular cartilage metabolism [36].Osteophyte maturity indicates the amount of bone tissue in osteophytes [37].OA is a whole-joint disease characterized by increased cartilage destruction, osteophyte formation, and SBP thickness [38].
As shown by Safranin O staining in Figure 3A, WGA-M001 had a suppression of cartilage destruction compared to the effect of the oral administration of phosphatebuffered saline (PBS) as a control in DMM-induced OA mice.Oral administration of a small amount of WGA-M001 (up to 100 mg/kg) provided protection against cartilage destruction, whereas high doses (500 mg/kg) of T. erecta and O. basilicum showed suppression of cartilage destruction.This shows that the amount of WGA-M001 oral administration is less than that of the T. erecta and O. basilicum extracts for suppressing cartilage destruction.Therefore, WGA-M001 is more attractive for OA treatment than the use of single extracts.The OARSI grade, SBP thickness, and osteophyte maturity showed results similar to the cartilage destruction phenotype observed with Safranin O staining (Figure 3B-D).Boswellia and Perna canaliculus were used as positive controls.Boswellia and P. canaliculus are health functional foods commonly used for treatment of OA [39][40][41].For centuries, extracts from Boswellia have held a significant place in traditional folk medicine [42], gaining popularity among consumers as a remedy for arthritis [43].As shown in Figure 3, WGA-M001 had a protective effect against cartilage destruction, compared to Boswellia and P. canaliculus.This result demonstrated that a mixture of T. erecta and O. basilicum showed a combined effect, resulting in cartilage recovery at lower concentrations compared to the recovery associated with each substance alone.As shown by Safranin O staining in Figure 3A, WGA-M001 had a suppression of cartilage destruction compared to the effect of the oral administration of phosphate-

WGA-M001 Attenuates OA Pathogenesis through Effective Gene Regulation with Respect to ERK and NF-κB Signaling
To demonstrate that we demonstrated that WGA-M001 has an inhibitory effect on catabolic factor expression, compared to T. erecta and O. basilicum treatment through inhibition of ERK and NF-κB pathway, RNA-seq analysis was performed to examine the regulation of the signaling pathways.NF-κB and MAPK (ERK, Jun N-terminal kinase (JNK), and p38) signaling pathways were primarily associated with the IL-1β signaling pathway [7].In silico analysis was performed to determine the pathways through which T. erecta, O. basilicum, and WGA-M001 regulate OA.Alterations in the expression patterns of genes related to each pathway mediated by WGA-M001, T. erecta, and O. basilicum were analyzed.A list of genes involved in each signaling pathway was derived using Ingenuity Pathway Analysis (IPA) [44].Alterations in expression patterns are presented as a heatmap (Figure 4A).Only genes related to each pathway whose expression was upregulated by IL-1β have been presented.Moreover, the expression patterns of WGA-M001, T. erecta, and O. basilicum were analyzed.Among the genes whose expression increased in the presence of IL-1β, we analyzed the number of genes whose expression increased to a greater degree following co-treatment with WGA-M001 compared to the increase associated with treatment with T. erecta and O. basilicum (Figure 4A).A total of 57 out of 72 genes related to the NF-κB signaling pathway and 59 out of 81 genes associated with ERK signaling were downregulated by WGA-M001, compared to the expression levels associated with other substances (Figure 4A).The results suggested that WGA-M001 blocked NF-κB and ERK signaling pathways, but not the other signaling pathway.We also investigated whether protein expression levels correlated with the heatmap outcomes (Figure 4B).As shown by the heatmap, the protein level of NF-κB and pERK was downregulated in the presence of WGA-M001 to a larger extent than the downregulation of the others (Figure 4C).
Thus, these data indicate that the effect of WGA-M001 is a synergistical effects of T. erecta and O. basilicum, and inhibits catabolic factor expression through downregulation of the NF-κB and ERK signaling pathways (Figure 6).

WGA-M001 Contains Quercetagetin-7-O-β-D-Glucopyranoside, Chicoric Acid, and Rosmarinic Acid
High-performance liquid chromatography (HPLC) experiments were performed to confirm the presence of phytochemical compounds in WGA-M001 (Figure 5).Comparative analysis using HPLC with standard compounds showed that Quercetagetin-7-O-β-Dglucopyranoside, Chicoric acid, and Rosmarinic acid were the most abundant in the sample treated with WGA-M001, and were detected at retention times of 16.6, 27.1, and 31.5 min, respectively.Thus, these data indicate that the effect of WGA-M001 is a synergistical effects of T. erecta and O. basilicum, and inhibits catabolic factor expression through downregulation of the NF-κB and ERK signaling pathways (Figure 6).Thus, these data indicate that the effect of WGA-M001 is a synergistical effects of T. erecta and O. basilicum, and inhibits catabolic factor expression through downregulation of the NF-κB and ERK signaling pathways (Figure 6).

Discussion
Owing to an increase in the elderly population and prevalence of obesity, the number of patients with OA is continuously increasing [45].Symptoms such as joint pain, stiffness, swelling, and tenderness significantly affect the patient's quality of life [46,47].Therefore, there is a significant need for preventing the onset of OA and alleviating its symptoms.It is well known that the pro-inflammatory cytokine IL-1β increases in the cartilage of patients with OA.IL-1β activates downstream signaling pathways like NF-κB and MAPK in the cartilage [48].NF-kB is an important transcription factor involved in immunity, inflammation, cell differentiation, and cell survival [49].In OA, IL-1β induces NF-κB activation through IκB degradation and p65 phosphorylation, leading to the upregulation of catabolic factors like MMP3 and MMP13, as well as an increase in inflammation-related molecules such as COX-2 and IL-6 [49].Similarly, the MAPK signaling pathway, consisting of three families (ERK, p38, and JNK), is involved in cell proliferation, inflammation, apoptosis, and differentiation [50].Activation of this pathway leads to the upregulation of catabolic factors such as MMP3 and MMP13 as well as an increase in inflammation-related molecules such as COX-2 and IL-6 [23,51,52].
Accordingly, many researchers continue their efforts to identify safe and sustainable drugs that can inhibit these molecules, and there is growing interest in natural products for this purpose [19].T. erecta has been studied for its ability to regulate NF-κB and p65 signaling in gastric cancer, induce anti-inflammatory responses, and reduce IL-6 levels [53].In chronic inflammatory conditions such as ulcerative colitis, T. erecta has shown effectiveness by reducing TNF-α and IL-6 levels [29].Similarly, O. basilicum has been studied for its ability to regulate NF-κB signaling in adipocytes, inducing anti-inflammatory responses by controlling IL-6, IL-1β, and TNF-α [54].Moreover, when T. erecta and O. basilicum were assessed via HPLC analysis, Quercetagetin-7-O-β-D-glucopyranoside was mainly present in T. erecta, whereas Chicoric acid and Rosmarinic acid were found in O. basilicum (data were not shown).However, WGA-M001 contained Quercetagetin-7-O-β-D-glucopyranoside, Chicoric acid, and Rosmarinic acid.The ratio of T. erecta to O. basilicum was 10:1.This combination may show a stronger inhibitory effect on WGA-M001 compared to that of T. erecta and O. basilicum total extracts.The HPLC data for WGA-M001 showed several peaks in a time-dependent manner.Although we have not characterized all the single compounds, they could be valuable single compounds with suppression of catabolic factor expression and cartilage destruction.In the near future, we will isolate each compound and characterize its inhibition effect on catabolic factor expression and cartilage destruction.These findings suggested that the two substances may have significant effects on OA.
In this study, the effect of T. erecta, O. basilicum, or a combination of the two, WGA-M001, to alleviate the pathogenesis of OA was investigated.Although T. erecta, O. basilicum, and WGA-M001 suppressed catabolic factor expression without cytotoxicity in chondrocytes, WGA-M001 had an additional inhibitory effect with respect to the catabolic factor expression through inhibition of gene regulation related to the ERK and NF-κB signaling pathway, compared to the effect of T. erecta and O. basilicum.Moreover, in DMM-induced OA mice, oral administration of WGA-M001 at doses up to 100 mg/kg effectively inhibited cartilage destruction compared to that associated with a high dose (500 mg/kg) of T. erecta or O. basilicum.Recent research has shown that Boswellia and P. canaliculus are well-known OA-alleviating substances [41,55,56].However, our study demonstrated that WGA-M001 had a stronger protective effect against OA development compared to the effect of the positive controls.Therefore, our in vitro, in vivo, and in silico analyses collectively suggest that WGA-M001, a mixture of both T. erecta and O. basilicum, may become a new treatment approach instead of Boswellia and P. canaliculus for OA and cartilage protection.

Sample Treatment and Reagents
T. erecta, O. basilicum, and WGA-M001 samples were supplied by the Wooree Green Science Corporation (Ansan, Korea).Dried T. erecta and O. basilicum was extracted using 50% EtOH for 24 h at 25 • C. The extracts were filtered using a Buchner funnel and concentrated using a rotary evaporator to obtain a 50% EtOH extraction powder.The 50% ethanol extract was fractionated using ethyl acetate and concentrated using a rotary evaporator.The dried samples were then stored at −80 • C in the refrigerator until further use.WGA-M001 is a mixture of two herbal extracts, i.e., T. erecta 50% ethanol extract powder and O. basilicum 50% ethanol extract powder in a ratio of 10:1.Samples were dissolved in dimethyl sulfoxide (DMSO) and boiled at 55 • C for 15 min.Human IL-1β recombinant protein (Z02922-10; GenScript, Piscataway, NJ, USA) was dissolved in PBS with 0.1% BSA.Twelve hours prior to cell harvesting, the samples (50, 100, and 200 µg/mL) were used along with IL-1β for co-treatment of mouse articular chondrocytes.

Primary Culture of Mouse Chondrocytes and Experimental Animals
Articular chondrocytes were isolated from the femoral condyles and tibial plateaus of 5-day-old ICR mice (DBL; Chungcheongbuk-do, Eumseong, Republic of Korea).Chondrocytes were isolated from cartilage tissues using Type II collagenase as previously described [57,58], and cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Capricon, Ebsdorfergrund, Germany) and 1% penicillin/streptomycin. Male C57BL/6J mice (10 weeks old) were purchased from DBL (Chungcheongbuk-do, Eumseong, Republic of Korea).All in vitro experiments were performed at least four times.All the experimental protocols were approved by the Animal Care and Use Committee of the University of Ajou (approval number: IACUC 2016-0041).The mice were housed at a temperature of 25 • C with a 12 h light/dark cycle.All mouse in vivo experiments were performed according to previously described protocols [59].

LDH Assay
The LDH assay was performed to measure the cytotoxicity of T. erecta, O. basilicum, and WGA-M001.Approximately 24 h before cell harvest, the medium was changed to 0% FBS medium and the cells were treated with T. erecta, O. basilicum, or WGA-M001 at 50, 100, and 200 µg/mL each.The LDH assay was performed using chondrocyte supernatants.To measure LDH activity, cytotoxicity was normalized to that of the untreated samples (0% cytotoxicity) and samples treated with Triton X-100 (100% cytotoxicity).Cell viability (%) was calculated using the formula 100 − (Sample OD − untreated sample OD)/(Triton X-100 OD − untreated sample OD) × 100.Absorbance was measured at 490 nm using a microplate reader (VICTOR X3; PerkinElmer, Waltham, MA, USA).

DMM-Induced OA Mouse Model and Oral Administration
For the experimental OA model, 10-week-old male C57BL/6 mice underwent DMM surgery using a previously described protocol [33,34].Each experiment was conducted independently at least five times.To perform DMM surgery, the fat pad was removed and the medial meniscus tibial ligament was incised.This method increased the mechanical stress in the knee joints of mice and led to cartilage degradation and osteoarthritis-like changes.After DMM surgery, mice were randomly divided into 10 groups: PBS, T. erecta (200 and 500 mg/kg), O. basilicum (200 and 500 mg/kg), and WGA-M001 (50, 100, and 200 mg/kg).Mice were orally administered PBS, T. erecta, O. basilicum, or WGA-M001 at different concentrations for 10 weeks [57,60].DMM-induced OA mice were orally administered Boswellia or P. canaliculus daily as positive controls.

Histological Analysis of Cartilage Destruction
DMM-induced OA mouse cartilage samples were obtained 10 weeks after DMM surgery, fixed in 4% paraformaldehyde overnight, and decalcified in 0.5 M EDTA for 2 weeks.The samples were then embedded in paraffin and the paraffin blocks were cut into sections of 5 µm thickness each.The sections were stained with hematoxylin, Safranin O, and fast green [33,34].The histological results were scored using the OARSI (Osteoarthritis Research Society International) grade, osteophyte maturity grade, and subchondral bone plate thickness method.A representative Safranin O staining image was selected from serial sections displaying the most advanced lesions.Three observers were blinded to the experimental group which evaluated the OARSI scores.The OARSI scoring system comprises seven grades (0-6).Grade 0 indicates a joint with no signs of osteoarthritis.Grades 1-6 signifies different levels of abnormality in the cartilage matrix and the presence of cells in the superficial zone.Specifically, they correspond to surface discontinuity, vertical fissures, erosion, denudation, and deformation [44,61].

HPLC Analysis of WGA-M001
HPLC analysis of WGA-M001 for chemical characterization and quantitative analysis of the selected compounds (Quercetagetin-7-O-β-D-glucopyranoside, Chicoric acid and Rosmarinic acid) were carried out on an Agilent 1260 Infinity II with diode array detector (Agilent Technologies, Santa Clara, CA, USA).The sample containing WGA-M001 was dissolved in 50% EtOH and filtered through a 0.45 µm nylon membrane filter.The components were separated using a Capcell Pak MG II C18 column (4.6 × 250 mm, 5 µm), with a column

Figure 4 .
Figure 4. WGA-M001 regulated OA-related molecules through dephosphorylation in the NF-κB and ERK pathways.For in silico analysis, 200 µg/mL of Tagetes erecta, Ocimum basilicum, or WGA-M001 was used for treatment of chondrocytes for 12 h along with IL-1β (1 ng/mL), and RNA sequencing was performed.(A) In each signaling pathway, the number of genes upregulated by IL-1β and then downregulated by T. erecta, O. basilicum, or WGA-M001 was shown.(B,C) Protein levels of pp38, p38, pJNK, JNK, pERK, ERK, pp65, p65, and IκB were detected by Western blot analysis and relative intensities were quantified by densitometry (n = 5).Each protein level was normalized to ERK.Data are represented as mean ± SD as results of analysis by one-way ANOVA with Dunnett's multiple comparisons test (n = 5).* p < 0.05, **** p < 0.0001, and n.s = not significant.

Figure 4 .
Figure 4. WGA-M001 regulated OA-related molecules through dephosphorylation in the NF-κB and ERK pathways.For in silico analysis, 200 µg/mL of Tagetes erecta, Ocimum basilicum, or WGA-M001 was used for treatment of chondrocytes for 12 h along with IL-1β (1 ng/mL), and RNA sequencing was performed.(A) In each signaling pathway, the number of genes upregulated by IL-1β and then downregulated by T. erecta, O. basilicum, or WGA-M001 was shown.(B,C) Protein levels of pp38, p38, pJNK, JNK, pERK, ERK, pp65, p65, and IκB were detected by Western blot analysis and relative intensities were quantified by densitometry (n = 5).Each protein level was normalized to ERK.Data are represented as mean ± SD as results of analysis by one-way ANOVA with Dunnett's multiple comparisons test (n = 5).* p < 0.05, **** p < 0.0001, and n.s = not significant.

Figure 6 .
Figure 6.The graphical summary of WGA-M001 illustrates its role in alleviating OA-related catabolic molecules through the inhibition mechanism of ERK and NF-κB signaling pathways.

Figure 6 .
Figure 6.The graphical summary of WGA-M001 illustrates its role in alleviating OA-related catabolic molecules through the inhibition mechanism of ERK and NF-κB signaling pathways.

Figure 6 .
Figure 6.The graphical summary of WGA-M001 illustrates its role in alleviating OA-related catabolic molecules through the inhibition mechanism of ERK and NF-κB signaling pathways.