JWPH International Journal Of Wellness And Public Health

1. Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammatory cell infiltration and bone destruction [1, 2]. Effector CD4+ T (Teff) cells, such as regulatory T (Treg) cells and other T helper cells (Th1 and Th17), are crucial in controlling the process of RA [3, 4].

Importantly, the Th17/Treg imbalance contributes to the pathogenesis and progression of RA [5, 6]. Therefore, targeting Th17 cells or combined with immune suppression can exert a therapeutic effect on RA [7]. Currently, the main treatments for RA are nonsteroidal anti-inflammatory drugs (NSAIDs); however, about 30%-50% of RA patients do not response to the treatment well [8]. Therefore, developing novel candidates is needed to meet the huge demand.

Carnosol is an orthodiphenolic diterpene stemmed from rosemary (Rosmarinus officinalis) and mountain desert sage (Salvia pachyphylla) [9]. In structure, carnosol has an abietane carbon skeleton with hydroxyl groups at positions C11 and C-12 and a lactone moiety across the B ring [10].

The special hydroxyl groups endow carnosol with a unique function of antioxidant [10]. Studies confirmed that treatment with carnosol suppressed interferon-γ- (IFN-γ-) induced inflammatory responses and hydrogen peroxide-
(H2O2-) induced oxidative stress in several types of cancer cells [11]. Carnosol also increases the Nrf2 binding with
Hindawi BioMed Research International Volume 2023, Article ID 1179973, 10 pages https://doi.org/10.1155/2023/1179973 ho-1-ARE, thereby contributing to HO-1 expression [11].

Moreover, it also promotes intracellular level of glutathione (GSH) and GSH synthesizing enzyme glutamate cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) [10]. Moreover, it also has antioxidant activity.

Inflammation induces oxidative stress, resulting in accumulation of reactive oxygen species (ROS), prostaglandins (PGs), cyclooxygenase2 (COX-2), interleukins (IL), and chemokines, which aggravate tumorigenesis or autoimmune inflammation [12].

Carnosol was verified to attenuate the expression of iCOX-2 and the production of PGE2 in both murine macrophage
and human mammary epithelial cells [13].

From the perspective of clinical translation, carnosol has the potential to treat various types of diseases. It has been
documented that carnosol was capable in treating LPSinduced septic shock in mice by suppressing inflammasome
activation [14]. Additionally, it was effective in treating numerous types of cancers, such as lung cancer [15], colon cancer [16], breast cancer, pancreatic cancer [17], prostate cancer [18], leukemia [19], and brain cancer [20].

Also, carnosol can protect against renal ischemia-reperfusion injury [21] and spinal cord injury [22]. Importantly, carnosol was reported to suppress AIDs, such as ovalbumin-induced allergic asthma [23] and experimental autoimmune encephalomyelitis (EAE) [24].

Li et al. firstly reported that carnosol and rosmanol synergistically alleviate murine arthritis via inhibiting TLR4/NF-κB/MAPK pathway [25].

Given the essential role of Th17 and Treg cell subsets in arthritis, we are wondering whether carnosol can modulate the differentiation and/or function of these cells that contributes to the therapeutic effects on CIA.

In this study, we observed that the administration of carnosol dramatically alleviated the severity of CIA model with
a decreased clinic score and joint inflammation. Remarkably, the frequency of Th17 cells was markedly reduced, which was positively correlated with the duration of joint inflammation. Besides, carnosol can also maintain the suppressive capacity of Treg cells in CIA model. In vitro, we confirmed that carnosol inhibits the IL-6R (CD126) expression that can suppress Th17 cell differentiation and promote Treg cell differentiation. Collectively, our results suggest that carnosol can alleviate the severity of CIA via hiding Th17 cell differentiation and maintain the stability of Treg cells; administration of carnosol can be applied as a potential therapy for patients with RA.

2. Results

2.1. Carnosol Alleviates the Development of CIA by Suppressing Lymphocyte Infiltration.

To investigate the therapeutic effect of carnosol in the context of autoimmune arthritis, we developed CIA model as previously described [26]. Carnosol was intraperitoneally administrated into the mice every other day after immunization for 14 continuous times at a dose of 50 mg/kg. We observed a significant attenuation in arthritis clinical scores and an obvious delay in CIA onset compared to DMSO administration (p < 0:01) (Figure 1(a)). Also, both the lymphocyte numbers in spleen and draining lymph nodes (dLN) were much decreased in carnosol-treated mice, compared with the DMSO group (p < 0:01) (Figure 1(b)). Accordingly, joint H&E staining was applied. Histological changes in the whole ankle joints demonstrated a significant decrease in synovitis, pannus formation, and destruction of bone and cartilage after treatment with carnosol (p < 0:05) (Figures 1(c) and 1(d)). Together, these results demonstrate that administration of carnosol has a robust therapeutic effect in murine experimental arthritis mice.

2.2. Carnosol Reduces the Frequency of Th17 Cells and Local Joint Inflammation in CIA Model.

As mentioned above, T helper cells are the most important proinflammatory response involved in the pathology of RA/CIA. We hypothesized that carnosol could have an impact on these effector cells. Next, we analyzed the frequencies of Th1, Th17, and Treg cells in carnosol- and DMSO-treated mice individually. As expected, the analysis clearly demonstrated that the number of Th17 cells was markedly declined in the dLN of CIA mice on day 54 compared to model or DMSO group (p < 0:01) (Figures 2(a) and 2(c)). Although the frequency of Th1 cells in carnosol treatment group was not statistically significant, it also showed a decreasing trend to some extent (Figures 2(a) and 2(c)). Meanwhile, our results revealed that carnosol was also able to induce Treg responses, which the frequency showed a slight increase, but no difference between model and vehicle group (Figures 2(b) and 2(c)). As is well known, Th17 cells play a driving role in the development of RA by altering the ratio of Treg/Th17 cells [27]. We therefore regressed the correlation between inflammatory scores (Figure 1(d)) and Th17 cell frequency (Figure 2(a)). Consistent with others, we also confirmed that Th17 frequency was positively correlated to inflammatory score in CIA mice (Figure 2(d)). Collectively, these results indicate that carnosol alleviates CIA via suppressing the differentiation of Th17 cells. Furthermore, we investigated the proinflammatory cytokines and Foxp3 levels in local joints using qRT-PCR analysis. The results revealed that, consistent with the Th17 frequency changing, the mRNA level of IL-17A was significantly decreased in carnosol-treated joint tissues in comparison to model or vehicle group (p < 0:05) (Figure S1A, left panel). Although Th1 cells in carnosol-treated CIA mice were not statistically decreased, the mRNA level of IFN-γ is lowered with statistical significance in local joint (p < 0:05) (Figure S1A, middle panel). However, Foxp3 mRNA in carnosol group was not significantly changed (Figure S1A, right panel). Interestingly, the level of IL-6 mRNA in carnosol-treated CIA joint tissues was significantly decreased (p < 0:01) (Figure S1B), implying that carnosol could inhibit joint erosion by suppressing IL-6 secretion. Thus, carnosol not only suppresses the differentiation of Th17 cells but also hides the transcriptional level of proinflammatory cytokines, such as IL-17A, IFN-γ, and IL-6, which contribute to the prevention of CIA progress.

Conclusion

In RA, the stable function of Treg cells plays a pivotal role in maintaining immune microenvironment homeostasis [28]. Therefore, we explored the Treg cell stability in vivo 2 BioMed Research International using the established ex vivo experiment system. dLN CD4+ CD25+ Treg cells were sorted from carnosol- and DMSO-treated CIA mice individually. The purity was confirmed by flow cytometry (Figure 3(a)). As we had expected, CD4+ CD25+ IL-17A+ Treg cells in carnosoltreated group were statistically lower than those on the corresponding groups after being stimulated with recombinant murine IL-6 (rmIL-6) for 3 days (p < 0:05) (Figures 3(b) and 3(c)). These results indicate that carnosol prevents Treg cells from converting to Th17 cells, which may sustain their suppressive function in CIA inflammatory environment. In addition to stability, we also focused on whether carnosol treatment affects the Treg cell immunosuppression. Hence, dLN CD4+ CD25+ Treg cells were harvested from each group on day 54 after CII immunization, and then, a standard in vitro functional assay was performed in the presence of rmIL-6 (Figures 3(d) and 3(e)). The results showed that Treg cells from carnosol-treated CIA mice maintained their suppressive function, which showed a decreased CFSE proliferation, while Treg cells from model-derived or DMSO-treated CIA mice notably lost their suppressive capacity.

 

Do any authors have Conflicts of Interest to declare?

no

I have reviewed and understood all of the above.

yes