Baicalin Alleviates Silica-Induced Lung Inflammation and Fibrosis by

Nov 25, 2015 - Silicosis is an inflammatory and fibrotic lung disease caused by inhalation of silica. Th17 cells play a key role in causing silica-ind...
1 downloads 13 Views 5MB Size
Download PDF
Article pubs.acs.org/jnp

Baicalin Alleviates Silica-Induced Lung Inflammation and Fibrosis by Inhibiting the Th17 Response in C57BL/6 Mice Tao Liu,†,‡ Wujing Dai,† Chao Li,† Fangwei Liu,† Ying Chen,† Dong Weng,† and Jie Chen*,† †

Department of Occupational and Environmental Health, School of Public Health, and ‡Department of Natural Products Chemistry, School of Pharmacy, China Medical University, Shenyang 110122, People’s Republic of China S Supporting Information *

ABSTRACT: Silicosis is an inflammatory and fibrotic lung disease caused by inhalation of silica. Th17 cells play a key role in causing silica-induced lung inflammation and fibrosis. Baicalin, a compound isolated from the Chinese herb Huangqin, could suppress the differentiation of Th17 cells and alleviate inflammation. However, there are very few reports of the immunoregulatory mechanisms of baicalin in experimental silica-induced lung inflammation and fibrosis. In our study, mice were exposed to silica by intratracheal instillation, and in this way we established an experimental silicosis model. To elucidate the effects and mechanisms of baicalin in silicainduced inflammation and fibrosis, we used baicalin to treat the developed mouse model of silicosis. Treatment with baicalin attenuated the accumulation of inflammatory cells and led to milder pathological inflammatory and fibrotic changes in lung tissues. Baicalin affected the immunological balance between Th17 and Treg responses. Therefore, baicalin caused a decrease in Th17 cells by stimulating Treg cells and by inhibiting IL-6 and IL-23. We further demonstrated that silica-induced Th1 and Th2 immune responses were both inhibited by increased Treg activation, which was promoted by baicalin. Our findings confirmed the potential functions of baicalin in inhibiting the Th17 response and reducing silica-induced inflammation and fibrosis.

B

silicosis due to the depletion of CD4+CD25+ regulatory T cells.13 A substantial body of evidence suggests that nTregs and iTregs, which are two major subpopulations of Treg cells, contribute to controlling disease in a cytokine IL-10- and TGFβ-dependent manner.10 Studies have also shown that Treg cells can have suppressive effects on many Th1- and Th2-type diseases.14,15 There is a reciprocal balance between Treg cells and Th17 cells.16 Th17 cells represent a novel subset of the specialized Th cell lineage and have been shown to produce IL-17, in particular IL17A.17 Recently, IL-17A was reported to modulate early neutrophilic lung inflammation in experimental silicosis. Neutrophils were significantly decreased in IL-17A-null mice in the early stage of silicosis, and acute lung inflammation was clearly reduced in silica-treated IL-17R-deficient mice.18,19 RORγt is an exemplary transcription factor for transcriptional regulation of Th17 cells. Pulmonary expression of IL-23 and IL6, but not TGF-β, was concurrent with IL-17A production; however, for differentiation, Th17 cells have been shown to require TGF-β in combination with IL-6.20,21 IL-6 has been established as an essential differentiation factor for Th17 cells;22 another cytokine, IL-23, important in resistance to infection, can promote IL-17A production, and mice deficient in IL-23 may also lack Th17 cell expression.23−25 Blocking IL-17 could result in a rapid increase of Treg cells in the lymph nodes of mice.26 Inhibition of IL-17A could delay Th1/Th2 responses by

aicalin (5,6-dihydroxyflavone 7-O-β-D-glucuronide; molecular weight = 446.36) is a main flavonoid compound isolated from the roots of the Chinese herb Huangqin (Scutellaria baicalensis Georgi) and has multiple bioactive effects such as anti-inflammation, antibiosis, anticancer, and smooth muscle relaxant properties and liver protection activities.1,2 Studies have shown that baicalin could achieve the resulting anti-inflammatory effects by immunomodulatory mechanisms, namely, suppression of macrophage activation, proliferation of mononuclear cells, and inhibition of the expression of Th1 cell-related cytokines.2,3 Previous studies have also indicated that baicalin may suppress newly generated Th17 cells by reducing RORγt expression in vitro. In vivo, baicalin could inhibit Th17 cell differentiation and infiltration and protect MRL/Ipr mice against nephritis.1 In addition, evidence has shown that baicalin induced Foxp3 protein expression in cultured T cells and promoted regulatory T cell (Treg) cell differentiation and regulatory activity.4 Silicosis is caused by inhalation of silica and is one of the primary types of pneumoconiosis, as well as being a relevant occupational disease worldwide.5−7 It is characterized by interstitial inflammation and fibrosis8 and involves innate and adaptive immune responses. In the pathogenesis of silicosis, Th1 and Th2 polarization is one sign indicating that silicosis inflammation has entered the stage of fibrosis. In addition, Th1/Th2 balance can be modulated by Tregs.9−11 Treg cells function as immune suppressors by regulating other effector T cells via multiple molecular mechanisms that regulate immune system homeostasis.12 Our previous studies have shown that early lung inflammation could be enhanced in © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 30, 2015

A

DOI: 10.1021/acs.jnatprod.5b00868 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Baicalin reduced the accumulation of neutrophils and lymphocytes in silica-induced inflammation. Total cells (A), neutrophils (B), lymphocytes (C), and macrophages (D) in the BALF. Cells were counted using Giemsa staining (n = 3−5) (*compared with the saline group, p < 0.05; #compared with the silica group, p < 0.05).

promoting the function of Treg cells.27 Taken together, we can therefore conclude that Th17 cells and associated relevant cytokines could play a key role in the pathogenesis of silicosis. Controlling Th17 cells and the balance of Th17/Treg responses may be a promising therapeutic approach for chronic inflammatory injury of silicosis. Currently, many traditional Chinese medicines are used for the treatment of silicosis, but their mechanisms are still not clear.28−30 Monomeric compounds derived from natural products that apply to treat silicosis are seldom reported. The immunoregulatory mechanisms of baicalin in the progress of experimental silica-induced inflammation and fibrosis are poorly understood. We set up a murine model of silicosis to determine whether baicalin can alleviate silica-induced inflammation and fibrosis by controlling Th17 or Treg cell responses and to gain insight into the mechanisms involved in the effects caused by administering baicalin. In this study, we observed that baicalin was able to inhibit the Th17 response by promoting differentiation of Treg cells and suppressing the expression of IL-6 and IL-23 in vivo. In addition, stimulated Tregs could cause inhibition of Th1 and Th2 responses. Notably, inflammation and fibrosis caused by silicosis were indeed alleviated by baicalin in C57BL/6 mice. Baicalin is a principle of the well-known Chinese herb Huangqin. This study is important for clarifying its mechanisms in silicosis treatment and has guiding significance for finding other natural products with antisilicosis activities. Moreover, the results of this study are also significant for discovering new effects and adaptation diseases of baicalin.

need to be explored.8,13,31−33 In recent years, the use of components from medicinal plants has become a promising approach for therapeutic treatment in many inflammatory disorders. However, there are currently very few reports on the effect of baicalin on silica-induced lung inflammation and fibrosis. Baicalin has been demonstrated to possess antiinflammatory properties and exert therapeutic effects in various chronic inflammatory diseases. Evidence has shown that baicalin was able to inhibit inflammatory cell infiltration in arthritic mouse ankles. In an EAE (experimental autoimmne encephalomyelitis) model, baicalin also decreased mononuclear infiltration in the spinal cord.2,34 In our study, we have provided the first evidence that baicalin could reduce the severity of silica-induced lung inflammation and fibrosis by inhibiting the Th17 response. Baicalin Attenuated Silica-Induced Lung Inflammation and Fibrosis. First, we counted the number of total cells, macrophages, neutrophils, and lymphocytes in the bronchoalveolar lavage fluid (BALF) of mice treated with saline, silica, and baicalin+silica at days 7, 28, and 56. The number of total cells in the baicalin-treated group significantly decreased compared with the silica group at days 7, 28, and 56 (p < 0.05; Figure 1A). The number of neutrophils increased in the BALF after silica treatment at days 7 and 56, with a peak at day 7. Conversely, the number of neutrophils was significantly reduced in baicalin-treated mice compared with silica-treated mice at day 7 (p < 0.05; Figure 1B). Regarding lymphocytes, at days 7, 28, and 56, the number of cells all indicated a significant reduction in baicalin-treated mice compared with silica-treated mice (p < 0.05; Figure 1C). Although the levels of macrophages in the baicalin group were all lower than the silica-treated group at each time point, the differences between the silica- and baicalin-treated groups were not significant (Figure 1D). These results suggested that treatment of baicalin reduced the cell

■

RESULTS AND DISCUSSION Many therapeutic methods for silicosis have been proposed, but as yet, no curative therapies have been found. Therefore, novel and effective approaches for treating silicosis are needed. In addition, the mechanisms of lung inflammation and fibrosis still B

DOI: 10.1021/acs.jnatprod.5b00868 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

fibrotic thickening, which appeared in the lung tissues at day 7. At day 28, collagen deposition and cellular nodules were observed in the silica group. At day 56, there were more areas of fibrotic bands in the lungs of mice compared to the previous two time points in the silica group. Conversely, there was a significant reduction of lung fibrosis in the baicalin+silica group compared with the silica group at equivalent time points, especially days 28 and 56. Additionally, the level of silicainduced fibrosis lung in the baicalin+silica group gradually increased from day 7 to day 56. Our data showed that baicalin contributed to the inhibition of inflammatory cell infiltration and controlled the development of silica-induced lung inflammation. We counted the number of total cells, neutrophils, lymphocytes, and macrophages in the BALF. Among all the inflammatory cells, the accumulation of neutrophils and lymphocytes significantly decreased in the baicalin-treated group compared with the silica group at day 7 or each time point. Neutrophils, lymphocytes, and macrophages have important roles in the early stage of lung inflammation in response to silica particles. Some evidence has also indicated that lung tissue injury is mainly caused by the accumulation and activation of neutrophils.35 Our findings suggested that baicalin may limit the recruitment of neutrophils and lymphocytes at different stages, especially exerting an inhibitory effect on neutrophils at an early stage of lung inflammation. We subsequently observed pathological changes due to inflammation of lung tissues. After lung tissue exposure to silica, the infiltration of inflammatory cells was rapidly exacerbated, then gradually alleviated at middle and late stages, and alveolar septal thickening followed the same trend. Conversely, the mice in the baicalin+silica group showed less infiltrated inflammatory cells in the lungs at each equivalent time point. The infiltration levels of inflammatory cells were consistent with the enhancement of inflammatory cells in the BALF. These results indicated that baicalin attenuated silica-induced lung inflammation. Baicalin Inhibited the Th17 Response in SilicaInduced Lung Inflammation and Fibrosis. To examine the effect of baicalin on Th17 cells in silica-induced inflammation, we evaluated the expression of CD4+IL-17A+T cells in hilar lymph nodes (HLNs) and spleens. In HLNs, the percentage of CD4+IL-17A+T cells in the baicalin+silica group decreased significantly compared with the silica group at days 7 and 28 (p < 0.05; Figure 4A), but had almost the same level compared with the silica group at day 56. At days 7 and 28, the percentage of CD4+IL-17A+T cells in the silica group increased significantly compared with the saline group (p < 0.05; Figure 4A), but there was no significant difference between the two groups at day 56. In the spleen, the percentage of CD4+IL-17A+T cells in the baicalin+silica group decreased significantly compared with the silica group at days 7, 28, and 56 (p < 0.05; Figure 4B). At each time point, the percentage of CD4+IL-17A+T cells in the silica group increased significantly compared with the saline group. We then examined the level of IL-17A mRNA expression in lung by real-time reverse-transcription polymerase chain reaction (RT-PCR). IL-17A, the typical effecter cytokine of Th17 cells, decreased significantly in the baicalin group compared with the silica group at day 7 (p < 0.05; Figure 5). Similarly, RORγt mRNA expression in the spleen had dramatically lower levels in the baicalin group compared with the silica group (p < 0.05; Figure 5). In the lung, RORγt mRNA even showed a significant decrease in the baicalin group compared with the silica group at each time point (p < 0.05;

accumulation of neutrophils and lymphocytes in silica-induced inflammation. To further confirm the effect of baicalin in the pathogenesis of silica-induced lung inflammation, the lung tissues of mice were observed using light microscopy to monitor pathological changes. No obvious abnormalities were observed in the lungs of mice that received saline at days 7, 28, and 56 (Figure 2).

Figure 2. Baicalin attenuated silica-induced lung inflammation. A1− A3, day 7; B1−B3, day 28; and C1−C3, day 56. A1−C1, saline group; A2−C2, silica group; and A3−C3, baicalin+silica group. H&E staining (200×).

The lungs of mice in the silica group showed a greater infiltration of inflammatory cells and alveolar septal thickening compared with the saline group at the equivalent time point. There was a gradual reduction of inflammatory cell infiltration in the silica group from day 7 to day 56, but the level of alveolar wall thickening gradually increased from day 7 to day 56. At equivalent time points, the baicalin+silica group had less infiltration of inflammatory cells and alveolar wall changes compared with the silica group. Subsequently, we examined fibrotic changes in the lungs of mice by Masson staining (Figure 3). Pathological examination showed no fibrosis generated in the lungs of mice of the saline group at days 7, 28, and 56. In the silica group, there was some

Figure 3. Baicalin attenuated silica-induced lung fibrosis. A1−A3, day 7; B1−B3, day 28; and C1−C3, day 56. A1−C1, saline group; A2−C2, silica group; and A3−C3, baicalin+silica group. Masson staining (200×). C

DOI: 10.1021/acs.jnatprod.5b00868 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 4. Baicalin decreased Th17 cells in the silica-induced inflammatory response. The percentage of CD4+T cells that coexpressed IL-17A is shown in the graph (A, HLN; B, spleen; n = 5). Th17 cells in the HLN (C) and spleen (D) were calculated by flow cytometry (n = 5) (*compared with the saline group, p < 0.05; #compared with the silica group, p < 0.05).

Figure 5. Expression levels of IL-17A and RORγt mRNA in the lung or spleen were assayed by real-time RT-PCR using the −ΔΔCt method (n = 4, 5) (*compared with the saline group, p < 0.05; #compared with the silica group, p < 0.05).

and key transcription factor expression. We first found that, in flow cytometric (FACS) analysis, baicalin treatment led to a significant decrease of CD4+IL-17A+T cells in HLNs at days 7 and 28 and in the spleen at each time point. We then examined the gene levels of RORγt and IL-17A in the baicalin+silica group, the key transcription factor and major functional cytokine of Th17, respectively, by real-time RT-PCR. IL-17A mRNA expression in lung tissues was significantly decreased in the early phase of the silica-induced inflammatory response. RORγt could direct the differentiation program of Th17 cells.36 RORγt was also found to be drastically enhanced after treatment with silica particles in the lungs of mice in a murine model.18,19,32 Our study supported this evidence and showed that RORγt mRNA expression in spleen tissues had a similar trend to IL-17A expression. Therefore, baicalin may inhibit RORγt-mediated IL-17A expression in mice with silicosis.

Figure S1). RORγt is an essential transcription factor for the development and function of Th17 cells.36 These results suggested that baicalin could suppress the Th17 response. Th17 cells are induced from uncommitted CD4+T cells by a specific cytokine-driven signal pathway. The importance of Th17 cells and related cytokines in the pathogenesis of silicosis is believed to be definitive; or rather they exert a vital biological function in inflammation. IL-17A was shown to be required for early lung neutrophilic inflammation and acute tissue injury in an experimental model of silicosis. IL-17A may recruit leukocytes, in particular neutrophils, to the site of inflammation. Conversely, IL-17A neutralization may reduce neutrophil influx at the early stage of silica exposure in the lungs of mice.19,37 To investigate the effect of baicalin on the Th17 response in silicainduced lung inflammation, we examined corresponding aspects including cell frequencies, relevant cytokine secretion, D

DOI: 10.1021/acs.jnatprod.5b00868 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 6. Baicalin increased Treg cells in the silica-induced inflammatory response. The percentage of CD4+T cells that coexpressed Foxp3 is shown in the graph (A, HLN; B, spleen) (n = 4, 5). Treg cells in the HLN (C) and spleen (D) were calculated by flow cytometry (n = 4, 5) (*compared with the saline group, p < 0.05; #compared with the silica group, p < 0.05).

Figure S2). All relevant data showed that baicalin could promote the Treg/Foxp3 response during the entire process of silicosis. However, our previous studies demonstrated that Treg cells suppressed inflammatory cells in the early stage of silicosis.13 We therefore deduced that baicalin may act directly on Tregs, thus inhibiting the Th17 immune response. Tregs could inhibit Th1 and Th2 responses by affecting relevant cytokines and transcription factors. In order to ascertain this in the silicosis murine model, we examined IFN-γ and TNF-α expression to investigate Th1, and IL-4 and GATA-3 to investigate Th2 in order to determine the type of inflammation and fibrosis that was inhibited after treatment with baicalin. Analysis by real-time RT-PCR showed that there were slight differences in IFN-γ expression in lung tissues between the baicalin+silica group and the silica group at day 7, although in spleen tissues, IFN-γ mRNA expression decreased significantly in the baicalin+silica group compared with the silica group at days 7 and 28 (p < 0.05; Figure S3A and B). The percentage of CD4+T cells expressing IFN-γ in HLNs of the baicalin+silica group decreased significantly compared with the silica group at day 7 (p < 0.05; Figure S4). We subsequently measured the levels of TNF-α in the BALF using a cytometric bead array (CBA), and the level of TNF-α in the baicalin+silica group was significantly lower than the silica group at day 7 (p < 0.05; Figure S3C). Enhanced Treg expression by baicalin reduced the Th1 response in silica-induced lung inflammation in the mice model. Meanwhile, at days 7 and 56, the baicalin +silica group showed significantly lower GATA-3 mRNA expression in lung tissues compared with the silica group (p < 0.05; Figure S5). We then tested the levels of IL-4 in the BALF using CBA. Results showed that IL-4 levels in the BALF

Collectively, these results, which are consistent with other relevant reports,1,34 indicated that the Th17 response was inhibited due to treatment with baicalin in this mouse model of silicosis. Baicalin May Decrease Th17 Cells by Promoting the Treg Response. It is well established that the balance between Th17 and Treg cells plays a key role in the maintenance of immune homeostasis in vivo.23 To illustrate whether the effect of baicalin on Th17 cells could be connected to Tregs during silica-induced lung inflammation, we first examined the CD4+Foxp3+T-cell fractions in HLNs and spleens. In HLNs, the percentage of CD4+Foxp3+T cells in the baicalin+silica group increased significantly compared with the silica group at days 7, 28, and 56 (p < 0.05; Figure 6A). In the spleen, the percentage of CD4+Foxp3+T cells in the baicalin+silica group increased significantly compared with the silica group at each time point (p < 0.05; Figure 6B). Next, we tested Foxp3 expression in the lung and spleen by real-time RT-PCR. Foxp3 is a key transcription factor of Treg cells. The mRNA expression of Foxp3 in the silica group was greater compared with the saline group at all time points. In the lung, the level of Foxp3 mRNA expression in the baicalin group was greater compared with the silica group at each time point, especially at days 28 and 56 (p < 0.05; Figure 7), and in the spleen, baicalin caused the Foxp3 mRNA level to increase significantly at days 7 and 28 (p < 0.05; Figure 7). IL-10, a major effector cytokine of Tregs, showed significantly greater expression levels compared with the silica group at all time points in the lung, especially at days 7 and 28 (p < 0.05; Figure 7). In the lung, TGF-β mRNA expression in the baicalin group also increased compared with silica group at day 7 (p < 0.05; E

DOI: 10.1021/acs.jnatprod.5b00868 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

results suggested that baicalin promoted Treg cell differentiation in the mice model of silicosis. Foxp3, as a key transcription factor of Tregs, could restrict RORγt-mediated IL17 expression and Th17 cell differentiation.39 Our data showed that Foxp3 mRNA expression in lung tissues was also significantly improved by treatment with baicalin. IL-10 is one of the important cytokines with immune suppressive function. Tregs are also the main source of IL-10 in response to silica dust.13,40 In the real-time RT-PCR analysis, IL-10 and TGF-β mRNA expression in the baicalin+silica group was increased compared with that of the silica group at the early stage. This is consistent with the changes in Foxp3 mRNA expression. IL-10 and TGF-β should be affected by the level of Tregs instead of baicalin.41 These results indicated that baicalin may inhibit Th17 by promoting the Treg response. Moreover, this manipulation toward Th17 could occur in an IL-10-, TGFβ-, and/or Foxp3-dependent way in silica-induced inflammation and fibrosis. A previous study reported that baicalin could inhibit the Th1 response by suppressing dendritic cell maturation.42,43 However, some reports have shown that baicalin was unable to bind to and directly interact with IFN-γ and TNF-α.44 Our previous studies indicated that Treg cells may suppress the Th1 response and delay Th1/Th2 polarization toward a Th2 dominant response by secreting IL-10 and TGF-β in an experimental model of silicosis.13,18 In this murine silicosis model, IFN-γ mRNA expression and CD4+IFN-γ+T cell frequencies were dramatically decreased in the baicalin group at day 7. TNF-α protein expression was significantly lower in the baicalin group compared with the silica group at the early stage of inflammation. Up-regulated expression of IL-10, TGF-β, and Foxp3 is likely to be responsible for suppressing the Th1/IFN-γ response. There is a balance between the Th1 and Th17 responses,45 which results in a relatively weak suppressive effect to the Th1 response, exerted by baicalin. Finally, Th1/Th2 polarization was shown to be insignificant. Th2-type cytokine IL-4 and transcription factor GATA-3 in the baicalin+silica group were significantly inhibited compared with the silica group at different time points, which was also achieved by promoting the Treg response. The relatively slight pathological changes in lung fibrosis also verified that the Th2 response was weak at later time points after treatment with baicalin. Baicalin May Decrease Th17 Cells by Inhibiting the Expression of IL-6 and IL-23. To further investigate the effects of baicalin on the Th17 response in the silica-induced lung inflammation of mice, the expression of pro-inflammatory cytokines IL-6 and IL-23 in lung tissues was detected by realtime RT-PCR and CBA. IL-6 mRNA expression in lung tissues of the baicalin+silica group significantly decreased compared with the silica group at day 7 (p < 0.05; Figure 8). At the other two time points, there were no statistical differences in IL-6 mRNA expression between the baicalin group and the silica group. We tested the levels of IL-6 protein in the BALF using CBA. Similarly, BALF IL-6 levels in the baicalin group were significantly lower than the silica group at day 7 (p < 0.05; Figure 8). However, there was no significant difference at days 28 and 56. IL-23 mRNA expression was examined by real-time RT-PCR. IL-23 expression in the baicalin+silica group decreased significantly compared with the silica group at days 28 and 56 (p < 0.05; Figure 8), although IL-23 had a slightly greater expression than the silica group at day 7.

Figure 7. Expression levels of Foxp3 and IL-10 mRNA in the lung or spleen were assayed by real-time RT-PCR using the −ΔΔCt method (n = 4, 5) (*compared with the saline group, p < 0.05; #compared with the silica group, p < 0.05).

from the baicalin+silica group were lower than the silica group at each time point; in particular, there was a significant difference at day 28 (p < 0.05; Figure S5). These data suggested that baicalin greatly influenced the inhibition of the Th1 and Th2 responses by boosting Tregs in the process of silicainduced inflammation and fibrosis.38 Tregs and Th17 cells are reciprocally interconnected in their developmental programs. Substantial evidence indicates that Treg cells are capable of inhibiting the function of Th17 and other effector T cells and play a key role in inhibiting inflammation and preventing autoimmunity.4,9 Our previous studies showed that the neutralization of IL-17A stimulated Tregs in the early phase of the silica-induced inflammatory response.18 We therefore deduced that, if baicalin can enhance Treg function, it must also indirectly suppress the Th17 response. In this investigation, FACS analysis indicated that, after baicalin was administered intraperitoneally every day, the CD4+Foxp3+T cells of the spleen and HLNs all had a higher percentage of total CD4+T cells at days 7, 28, and 56. These F

DOI: 10.1021/acs.jnatprod.5b00868 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

tion.24,25,50 Together, our results suggested that Th17 cell maturation could be affected by baicalin-inhibited IL-23 expression at the middle and later stages of silica-induced inflammation and fibrosis, although baicalin did not affect the expression of IL-23R.1 In summary, we deduced that baicalin inhibited the Th17 response by suppressing IL-6 and IL-23 in the inflammation of silicosis, which could provide direct evidence of the baicalininduced inhibition of the Th17 response. Altogether, our studies suggest that baicalin alleviates silicainduced lung inflammation and fibrosis. Baicalin may play an antisilicosis function in mice by means of inhibition of Th17 cell differentiation, by promoting the Treg immune response and suppressing the pro-inflammatory cytokines IL-6 and IL23. Stimulation of Tregs also attenuated the Th1 and Th2 immune responses by inhibiting the expression of IFN-γ, TNFα, GATA-3, and IL-4. Additionally, this is a new report of in vivo clarification of the immunoregulatory mechanisms of baicalin in experimental silicosis and also confirms immunosuppressive activities of natural products aiming at Th17 in this murine silicosis model. Our results have suggested that baicalin may be a promising therapeutic compound for Th17 cellmediated silica-induced lung inflammation and fibrosis.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Natural crystalline silica particles (Min-U-Sil 5 ground silica; size distribution: 97%