Effects of a herbal formulation, KGC3P, and its individual component, nepetin, on coal fly dust-induced airway inflammation

Coal fly dust (CFD)-induced asthma model is used as an ambient particulate matter model of serious pulmonary damage. We aimed to evaluate the effects of a combination of ginseng and Salvia plebeia R. Br extract (KGC-03-PS; KG3P) and its individual components (hispidulin, nepetin and rosmarinic acid) in a CFD-induced mouse model of airway inflammation (asthma). We also evaluated signal transduction by KG3P and its individual components in the alveolar macrophage cell line, MH-S cells. In vitro, KG3P and its individual components inhibited nitric oxide production and expression of pro-inflammatory mediators and cytokines (iNOS, COX-2, IL-1β, IL-6 and TNF-α) through the NF-κB and MAPK pathways in coal fly ash (CFA)-induced inflammation in MH-S cells. Moreover, in the CFD-induced asthma model in mice, KG3P and its predominant individual component, nepetin, inhibited Asymmetric Dimethyl arginine (ADMA) and Symmetric Dimethyl arginine (SDMA) in serum, and decreased the histopathologic score in the lungs. A significant reduction in the neutrophils and immune cells in BALF and lung tissue was demonstrated, with significant reduction in the expression of the pro-inflammatory cytokines. Finally, IRAK-1 localization was also potently inhibited by KG3P and nepetin. Thus, KG3P extract can be considered as a potent candidate for amelioration of airway inflammation.

Inhibitory effects of KG3P and nepetin on serum asymmetrical dimethyl arginine (ADMA) and symmetric dimethyl arginie (SDMA) levels, and restoration of histopathological lesions. Asymmetrical dimethyl arginine (ADMA) and symmetric dimethyl arginie (SDMA) are involved in the inflammation, endothelial dysfunction and oxidative stress. Basically they are the structural analogues of l-arginine, which competitively regress NO synthase, ultimately leading to decreased basal NO production with the fact that basal NO production is essential for cellular proliferation, vasodilation and migration [18][19][20] . Therefore we had checked the effects of the KG3P treatment on the serum ADMA and SDMA levels. As shown in Fig. 3B,C, both ADMA and SDMA were potently reduced by the positive control, montelukast, and by higher doses of KG3P and nepetin. As observed in Fig. 3D,E, higher doses of KG3P and nepetin restored the histology of lungs toward normal and decreased the histopathological score.

Decreased number of immune cells in BALF and lung tissue. Generally, there is an increase
in immune cells during the invasion of foreign particles in the body which is the natural adaptive immune response 21 . We therefore sought to check the immune cell levels in the lungs and BALF. As shown in Fig. 4A-D, montelukast, both doses of KG3P, and nepetin potently suppressed the number of total immune cells and neutrophils in BALF and lung samples. Moreover, using FACS analysis (Table 1 and Fig. 5, A-G), CD4 + , CD8 + , and CD11b + cells were significantly decreased in BALF and lungs cells, indicating that the over activation of the immune system caused by CFD was positively suppressed by the higher doses of KG3P and nepetin, decreasing the aggravation of inflammation.

Suppression of pro-inflammatory cytokines expression in BALF and lung tissue.
Pro-inflammatory cytokines are secreted in response to inflammation and timely control is essential; this is because an uncontrolled secretion of these chemicals can irreversibly damage the tissue 22 . And it is a renowned fact that the major contributors in the pathology of asthma are the cytokines that are released from lung cells. Keeping this in mind, we had investigated the levels of these pro-inflammatory cytokines in the BALF and lung tissue. From Fig. 6A-C, the higher doses of KG3P and nepetin are shown to significantly suppress the levels of IL-17,which is naturally elevated in asthma causing allergic rhinitis 23 , TNF-α, which is found to be potently involved in many aspects related to airway pathology in asthma and which is also one of the target for asthma treatment 24 , and MIP2 which is activated by IL-17 25 . In addition to these, CXCL-1 which attracts neutrophils to the sites Scientific RepoRtS | (2020) 10:14036 | https://doi.org/10.1038/s41598-020-68965-5 www.nature.com/scientificreports/ of airway inflammation 26 in BALF samples from mice was also downregulated by KG3P treatment (Fig. 6D). Moreover, montelukast, both doses of KG3P, and nepetin significantly inhibited the expression of IL-17, IL-1β, IL-6 and TNF-α which are considered to be the top prioritized pro-inflammatory agents 27 ( Fig. 6E-H). Lastly, CCR3 which is present on the T-cells co-localizing with eosinophils in the allergic asthma condition 28 , and MUC5AC which is the major component of mucous causing airway obstruction in asthma 29 (Fig. 6I,J) were also ameliorated by KG3P and nepetin, indicating that the extract and the individual component are indeed potent anti-inflammatory agents.
Inhibition of fluorescence intensity of IRAK-1 and signal transduction of KG3P and nepetin via the NF-κB and MAPK pathways. Interleukin 1 receptor associated kinase (IRAK-1) is the negative regulator of Toll like receptor 1 and it is key activator of NF-κB and MAPK pathways. In fact, continuous activation of IRAK-1 causes the onset of persistent asthma in humans 30 . We therefore checked lung tissues which were stained with IRAK-1 antibody to visualize the presence of IRAK-1 positive cells with KG3P and nepetin. As shown in Fig. 7A,B, IRAK-1 positive cells were potently inhibited in the lung tissue by montelukast, both doses of KG3P, and nepetin. This was also confirmed via western blot analysis of lung tissue protein where the higher dose of KG3P and nepetin potently inhibited the expression of IRAK-1, p-TAK1, p-NF-κB, p-ERK, and p-JNK (Fig. 7C,D). These results strongly suggest that KG3P and the single compound, nepetin, exhibit their anti-inflammatory effects via the NF-κB and MAPK pathways during CFD-induced airway inflammation.

Discussion
Numerous epidemiological studies are available on the effects of fine and ultrafine particulate matter (PM) on health, especially those relating to pulmonary disorders that in the long-term, lead to extensive morbidity and mortality. The composition of PM include coal, ash, oil, and diesel exhaust particles (DEP) 31,32 . The incidence of www.nature.com/scientificreports/ chronic pulmonary diseases, especially lung cancer, that occur due to chronic deposits of these fine particles in tissues over a long period of time, is a major concern of scientists 2 . The current lifestyle trend inevitably exposes living organisms to the inhalation of these particles; thus, many scientists are attempting to devise various preventive and therapeutic remedies (of chemical and herbal origin) to avoid incidence of PM-induced chronic diseases. Few studies exist on the effects of natural herbal extracts or compounds on CFD-induced airway inflammation using a mice model. However, several studies have been performed using lung epithelial cells to examine the effects of CFD on reactive oxygen species or pro-inflammatory cytokines 33 . A study reported that iron, which constitutes up to 14% of CFA, was responsible for the expression and secretion of the pro-inflammatory cytokine, IL-8, in A549 human lung epithelial cells 34 . Another study examined the initiation and secretion of reactive oxygen species and cytokines, primarily IL-6, TNF-α, and IL-8 in human bronchial epithelial cells (BEAS-2B) and RAW 264.7 cells exposed to CFA or PMs 35 . Similar to this study, we found increased expression of NO, iNOS, COX-2, and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in the murine macrophage MH-S cell line when stimulated with CFA. However, as expected, these elevated levels of NO and pro-inflammatory mediators and cytokines were decreased by treatment with KG3P and its individual components ( Fig. 2A,B).
Based on the mechanistics, various studies have shown that these PMs can stimulate toll like receptor (TLR) 2 and 4. Once these particulate matters stimulate TLR4 receptors, the classical NF-κB pathway is mainly activated. This includes the activation of factors downstream of TLR4, which includes IRAK-1, transforming growth factor beta-activated kinase 1 (TAK-1), etc., which ultimately causes translocation of NF-κB from the cytoplasm to the nucleus to activate the NF-κB pathway 36 . Certainly, several studies have shown that in alveolar macrophage cells, treatment with a TLR4 antagonist results in minimal release of pro-inflammatory cytokines 37 . Importantly, in our study, CFA activated the NF-κB and MAPK pathways, both of which are primarily activated during foreign invasion, in this case the PMs relative to previously reported studies 38,39 . We hypothesize that CFA may bind to TLR4 receptors, as shown in Fig. 2C,D, as all downstream factors for NF-κB [mainly IRAK-1 and TAK-1, and MAPK (ERK, JNK and P38)] were activated by CFA and simultaneously inhibited by KG3P and the individual components. Among the individual components identified in the KG3P extract, nepetin was the most abundant. Previous reports on the biological activities such as the anti-microbial, anti-oxidant, anti-tumour, and antiinflammatory of nepetin have been presented; however, its effect on airway inflammation or asthma remained un-investigated [40][41][42] . Therefore, we continued our in vivo study with nepetin from the KG3P extract.
Many studies have reported the effects of either PMs, CFA, or CFD in various murine and rodent models of pulmonary ailments such as asthma and airway inflammation 43 . Walters et al. 44 reported that PMs induced an increase in the amount of immune cells in BALF and lung cells and increased the expression of pro-inflammatory www.nature.com/scientificreports/ cytokines in lung tissues. Takano et al. 45 reported that the inhalation of DEP exacerbates allergen-related eosinophil recruitment and airway hyper-responsiveness in mice. Similar to these studies, we found that exposure of mice to CFD resulted in elevated levels of ADMA and SDMA (both of which are analogues of NO during inflammation), causing histological changes in lung tissues including infiltration of inflammatory cells, collagenous fibre production, and increased mucous production due to downstream secretion of pro-inflammatory cytokines such as interleukins and TNF-α. Various immune cells in BALF and the amounts of neutrophils in lung tissues (Figs. 3,4,5) were positively inhibited by KG3P and nepetin, respectively. We also observed elevated expression of pro-inflammatory cytokines, primarily the interleukins (i.e., IL-17, IL-1β, IL-6, and TNF-α) (Fig. 6) in BALF and lung tissues; this is similar to previous studies that indicated the activation of pro-inflammatory cytokines during airway inflammation and our in vitro results using the CFAinduced MH-S cells 46 . Macrophage inflammatory protein-2 (MIP2), chemokine (C-X-C motif) ligand 1 (CXCL1), www.nature.com/scientificreports/ and CC-chemokine receptor 3 (CCR3) are major inflammatory factors expressed in airway inflammation and asthma [47][48][49][50][51] . In addition, polymeric mucin gene (MUC5AC) is expressed and its activation causes disruption in the secretion and storage of mucin, thus leading to mucus gel formation. This mucus gel obstructs the airway epithelium resulting in asthma and bronchitis 29,52 . In our mouse model, these factors were elevated; however, as expected, oral treatment with KG3P and nepetin significantly decreased the levels of these parameters, and the effects were comparable to those observed on treatment of mice with the positive control, montelukast (administered to treat asthma 53 ). This indicates that the decreased levels of expression of these genes inhibited the infiltration of inflammatory cells and decreased the thickness of the airway epithelium. We also confirmed the signal transduction of KG3P and nepetin via the IRAK-1-NK-κB pathway using immunohistofluorescence and immunoblot experiments. KG3P and nepetin potently inhibited the localization of IRAK-1 in lungs and inhibited the phosphorylation of IRAK-1, p-TAK-1 and NF-κB in lungs tissue, and ERK and JNK from the MAPK pathways (Fig. 7). These results confirm the in vitro data obtained using the CFA-induced MH-S cells. Therefore, we demonstrated the novel anti-inflammatory/anti-asthmatic effects of a herbal formulation with KG3P and those exhibited by its individual component, nepetin, on murine alveolar macrophage MH-S cells and CFD-induced airway inflammation mouse model. Using an even greater mechanistic study, this formulation may be proven to be a promising herbal remedy for the prevention of PM-/CFA-/DEP-/CFD-induced airway ailments. Values in bar graphs are expressed as mean ± SEM (n = 8 mice). # p < 0.05, ## p < 0.01, and ### p < 0.001 (compared to WT), and *p < 0.05, **p < 0.01, and ***p < 0.001 (compared to CTL).

Materials and methods
Chemicals and reagents. The detailed chemicals and reagents are given in Supplementary information 1.

Cell phenotypes in Lung, BALF
The instrumental conditions of UPLC-PDA were as follows: the chromatographic separation was obtained using a BEH C18 column (100 mm × 2.1 mm, 1.7 µm, Waters Co., USA) and the column temperature set to 40 °C. The binary gradient elution system consisted of 0.001% phosphoric acid in water (A) and 0.001% phosphoric acid in acetonitrile (B). Separation was achieved using the following protocol  Expression is presented as relative quantitation (RQ). Data are from individual mice, with arithmetic mean points shown in histograms. Values in bar graphs are expressed as mean ± SEM (n = 8 mice). # p < 0.05, ## p < 0.01, and ### p < 0.001 (compared to WT), and *p < 0.05, **p < 0.01, and ***p < 0.001 (compared to CTL). www.nature.com/scientificreports/ After extraction, the sample solution was filtered (0.2 µm, Acrodisk, USA) and injected into the HPLC-PDA system (Waters Co., USA). The instrumental conditions of HPLC-PDA were as follows: chromatographic separation was performed using a Zorbax eclipse XDB C18 column (250 mm × 4.6 mm, 5 µm, Agilent Co., USA). The binary gradient elution system consisted of 0.05% acetic acid in water (A) and methanol (B). Separation was achieved using the following protocol: Nitric oxide (NO) assay. The detailed method is given in Supplementary information 1.
Cell Viability (MTT) assay. The detailed method is given in Supplementary information 1.

RNA extraction and polymerase chain reaction (PCR).
The detailed method is given in Supplementary information 1 ( Table 2).

Western blot analysis.
The detailed method is given in Supplementary information 1. Following the acclimatization period, all groups except group 1 were administered 100 μL of CFD (Coal (5 mg/ml, Fly ash (10 mg/ml), and diesel exhaust particles (DEP; 5 mg/ml) in saline by intratracheal instillation thrice at 3 day intervals for 12 days using bronchial tubes, as previously described 54 . Montelukast, KG3P and nepetin were orally administered daily for 11 days at the above mentioned dosages. On day 12, all mice were euthanized and blood, bronchoalveolar lavage fluid (BALF), and lungs tissues collected for further experiments. The schematic diagram for the experimental protocol is presented in Fig. 3A.

Collection of bronchoalveolar lavage fluid (BALF) and lung cells. BALF was collected 24 h after
the last oral injection of samples. Mice were anesthetized by an i.p. injection of 10% urethane (100 µL; Sigma-Aldrich, Korea). A tracheotomy was then performed, and a cannula inserted into the trachea. Ice-cold DMEM was instilled into the lungs, and BALF collected. Total cell counts were measured with a haemocytometer. For cytological examination, cells were prepared with a Cytospin (Hanil Science, Korea), fixed, and stained with a modified Diff-Quick stain. Differential cell counts were determined using at least 500 cells on each cytospin slide. Blood was collected by cardiac puncture, allowed to clot, then centrifuged; aliquots of serum were stored at − 70 °C for ELISA.