The Effects of Nebulized Inhaled Triptolide on Airway Inflammation in a Mouse Model of Asthma

Inhalation of nebulized TP has received little attention in the past. Here, we intend to investigate the effect of nebulized inhaled TP on airway inflammation in a mouse model of asthma. 29 SPF BALB/c mice were divided into four groups: blank control (Blk, n = 5), normal saline (NS, n = 8), dexamethasone (Dex, n = 8), and TP (n = 8). During the process of sensitization, mice in the three intervention groups were treated with nebulized NS, an injection of Dex, and nebulized triptolide, respectively. Then bronchoalveolar lavage fluid (BALF), peripheral blood, and lung tissue were collected. Relevant cytokines, transcriptional factors, and CD4+Th17+ T cell proportions were assessed and compared. IL-6, IL-17, IL-23, and TGF-β1 demonstrated a significant difference between groups in the following order: Dex < TP < NS (P ≤ 0.001), while IL-10 changed in the opposite direction (P < 0.001). At the transcriptional level in lung tissue, the Ct value of IL-17 in the Dex group was significantly higher than in the NS and TP groups (P < 0.001). Meanwhile, it was higher in the TP group than in the NS group (P < 0.001). The Ct value of RORγt demonstrated a significant difference among three groups in the following order: Dex > TP > NS (P < 0.001). An opposite trend of FoxP3 Ct value was revealed in the order: NS > TP > Dex. The proportion of CD4+Th17+ cells was 9.53 ± 2.74% in the NS group, 4.23 ± 2.26% in the Dex group, and 6.76 ± 2.99% in the TP group, which shows significant differences between the NS and Dex (P < 0.001) or NS and TP groups (P < 0.05). Inhalation of nebulized triptolide can play a role in suppressing airway inflammation with inflammatory cytokines and transcriptional factors reduced and CD4+Th17+ T cells dampened, also in a manner less than injected dexamethasone.


Introduction
Among all asthma cases, approximately 10% are classifed as severe asthma.Current medication regimens, while generally efective, do not control symptoms for a subset of patients [1][2][3][4].Severe cases of asthma are often associated with refractory symptoms, glucocorticoid resistance, and death, at least 50% of which are accompanied by infltrated or increased neutrophils, rather than the eosinophilic phenotype [4][5][6][7][8].Previous studies have suggested that airway infammation dominated by neutrophil infltration plays a crucial role in such patients [9,10].Its mechanism is diferent from the typical, well-recognized T1/T2 imbalance and is more correlated with the imbalance of Treg/T17 lymphocytes [9,10], which is generally called a "non-type 2 immune response."Triptolide (TP) is a diterpene compound isolated from the thunder god vine Tripterygium wilfordii, which has signifcant anti-infammatory and immunoregulatory effects.Whether in the form of T. wilfordii polyglycoside or single extract, a large number of basic scientifc studies have suggested its therapeutic efect in many autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus [11][12][13].Titov and colleagues defned the molecular target of T. wilfordii, which lies in the RNA polymerase II (RNAPII) at the core of the transcription factor TFIIH subunits ERCC3 (i.e., XBP) [11].When TP binds to its target, the specifc efect on transcription results in broad inhibition of the immune system.Chinese investigators have also confrmed the anti-infammatory effects of TP in asthma [12][13][14][15][16][17].A cellular culture study showed that TP inhibited airway epithelial goblet cell proliferation and MUC5AC expression via the NF-κB pathway, while airway smooth muscle cell (ASMC) proliferation and migration induced by TGF-β1 were moderated through the Smad and NF-κB pathways, suggesting TP's potential therapeutic efect on airway remodeling [12,14].In a precedent asthmatic mouse model, levels of white blood cells (WBCs), eosinophils (EOS), IL-17, and IL-23 in BAL were signifcantly decreased, with mRNA expression of IL-17 in lung tissue also reduced by TP treatment [15].In another study on human PBMC, the transcription and expression of IL-13 can be reduced by TP by inhibiting the binding of nuclear transcription factors GATA3 and NFAT1 to the promoter, with the efector CD23+ cells suppressed [16].Moreover, the expression of IL-12/IL-23 could be blocked by TP through activating the CCAAT/enhancer binding protein (C/EBP) in antigen presenting cells (APCs), which enhances the promoter of the common subunit P40 shared by IL-12 and IL-23 [17].Despite the above activities ofered, TP has obvious toxicity and side efects, such as reproductive toxicity, liver and kidney damage, and bone marrow inhibition, which greatly hamper its further clinical application.
Aerosol inhalation is a common mode of drug administration for respiratory diseases.Te potential application of nebulized TP in rodent asthma models has received limited attention [12,14,15].In this study, we evaluated the potential therapeutic efects of aerosolized TP in a mouse model of asthma.We investigated its efcacy by observing the anti-infammatory efect of this innovative dosing route, especially its impact on Treg/T17 lymphocytes.

Materials and Methods
In an animal laboratory of Crown Bioscience Taicang (approved by AAALAC), 29 SPF grade BALB/c female mice (6-8 weeks) were divided into four groups (anaesthetized with 3% pentobarbital at 50 mg/kg via intraperitoneal injection, 40 μl/each as average).5 mice served as no-treatment controls.Te asthmatic models established were: 8 mice in the NS group, 8 cases in the Dex group, and 8 cases in the TP group.Ten normal saline (NS) atomization, dexamethasone (Dex) intraperitoneal injection, and aerosol TP (using an ultrasonic nebulizer) were administered.
24 mice were intraperitoneally injected with a 400 μl sensitizing solution of 10% ovalbumin (OVA, Sigma, Grade V, ≥98%) with 50 μl of Al(OH) 3 (aluminium hydroxide adjuvant) 1 mg at 0, 7, and 14 days, respectively.Ten 2 mg/ ml OVA-NS solution (20 drops each by nasal drip) was given once a day as a stimulus for 5 days continuously from the 21st day.Preceding the stimulation, treatment groups were kept in animal chambers and treated once per day with NS atomization (30 min), Dex intraperitoneal injection 2 mg/kg (250 μl as average), or nebulized TP (NSABE, purity >98%, not derivative, insoluble in water) 2 mg/ml in the solution consisting of double distilled (dd) H 2 O, 5% DMSO (dimethyl sulfoxide), and 30% PEG (polyethylene glycol) with a nozzle fxed upon the face of mice for 30 min.
Te mice were sacrifced within 24 hours after the fnal stimulation by an overdose of anaesthesia (3% pentobarbital at 200 mg/kg via intraperitoneal injection, 160 μl/each as average).Te trachea was intubated, and then the left main bronchus was ligated.NS 2.4 ml was used to perfuse the right lung tissue 3 times and then extracted.Bronchoalveolar lavage fuid (BALF) was obtained with a recollection amount of ≥80%.Te supernatant was then centrifuged at a speed of 1500 rpm at 4 °C for 10 min and frozen at −70 °C for measurement of IL-6, IL-10, IL-17, IL-23, and TGF-β by ELISA (BioSwamp Life Science Lab, China).
Part of the left lung tissue of mice was resected, fxed with 10% paraformaldehyde, followed by dehydration, parafn embedding and sectioning, haematoxylin eosin staining, and sealing.Te tissue morphology, infammatory infltration, and mucosal damage were then observed under a light microscope.
Total RNA was extracted from the tissue of the left lung with Trizol, reverse-transcribed into cDNAs, and used as templates for PCR amplifcation.Te target gene primer sequences are shown in Table 1.
Peripheral blood mononuclear cells (PBMC) were isolated from 1 ml of blood from each mouse, adjusted to a density of 1 × 10 6 /L, and added to a 24-well culture plate with RPMI-1640 culture medium.For each well, 1 ml of phorbol ester (PMA, 10 ng/ml), ionomycin (1 μg/ml), and monomectin solution (500 ng/ml) were added and placed in a 5% CO 2 incubator at 37 °C for 4 hours.
First, 0.5 μl of anti-IL-17 Ab was added to the cell suspension, followed by repeated centrifugation, then removing the supernatant, washing and resuspension of the cell pellet, and then incubation at 4 °C for 1 hour.After washing with PBS solution twice, fow cytometry (FCM) was used to detect the percentage of T17+ T cells among CD4+ cells in peripheral blood (Accuri C6, BD, USA).
SPSS13.0 software and R3.6.1 were used for statistical analysis and relevant charts; ANOVA for comparison among groups; and t-test for pairwise comparisons (LSD adopted).Te measurement data showed as a mean-± standard deviation (SD), statistical inferences made by bilateral tests, and P < 0.05 was considered statistically signifcant.

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Measurement of Key Transcription Factors (TFs) in Lung
Tissue.∆Ct value of each TF was calculated by subtracting each Ct value from the reference GAPDH.Te ∆Ct value of IL-17 in the Dex group was signifcantly higher than that in the NS and TP groups (P < 0.001).Tere was also a signifcant diference between the latter two groups (P � 0.003), suggesting the mRNA level of IL-17 was the lowest in the Dex group (except the Blk group).Te ∆Ct value of RORct showed signifcant diferences in pairwise comparison, ranking as Dex > TP > NS group (P < 0.001), suggesting that mRNA expression of T17-specifc transcription factors was reduced by Dex and TP, with a greater efect of systemic Dex.Te ∆Ct value of FoxP3 in the NS group was signifcantly higher than that of both Dex and TP groups (P < 0.001), with a signifcant diference between the Dex and TP groups (P � 0.001), which displayed a lower level in the Dex group and the suppressive efect upon the transcription of FoxP3 mRNA (shown in Table 3 and Figure 2).Shown in Figure 3 are the graphs of fow cytometry on CD4+T17+ T lymphocytes in PBMC from samples of four groups.Te proportion of T17+ was 9.53 ± 2.74% in the NS group, 4.23 ± 2.26% in the Dex group, and 6.76 ± 2.99% in the TP group.A statistical diference existed between the NS and Dex groups (P < 0.001), indicating the inhibition of Dex on T17.It appeared that TP had an apparent downregulatory efect on T17 cells compared to NS-treated mice (P � 0.038).However, there was no signifcant difference between the TP and Dex group (P � 0.056) (shown in Table 3 and Figure 4).A signifcant pathologic diference among the NS group, the treated groups, and the blank group could be found in pairwise comparison (shown in Figure 5), while no signifcant diference was seen between the Dex group and TP group in terms of pathological scores on lung tissue (by a pathologist).

Discussion
Te results confrmed that nebulized TP has antiinfammatory activity in our mouse model of asthma.Proinfammatory cytokines such as IL-6, IL-17, IL-23, and TGF-β all decreased in BALF due to the efect of Dex or TP.Although the anti-infammatory efect of inhaled TP was not at the level of i.p. injected Dex, the trend of declining infammation was consistent.Te mRNA of IL-17 and RORct (the transcription factor that promotes IL-17 transcription and T17 cell diferentiation) decreased in both the Dex and TP groups (according to Ct values), while FoxP3 (the transcription factor that promotes Treg cell diferentiation) increased in both the Dex and TP groups.CD4+T17+ T cells isolated from PBMC were also reduced in numbers by Dex and TP.Te latter could induce the downward trend and reach statistical signifcance as Dex, though to likely a less extent.Pathological changes in lung tissue also verifed the mouse model of asthma and antiinfammatory efects of TP.Hence, our results provide evidence that TP could attenuate T17 expression and increase Treg expression simultaneously in this mouse model of asthma.
We are particularly interested in Treg/T17 cells which are important in the complex pathogenesis of bronchial asthma.Distinct from the traditional hypothesis of T1/ T2 imbalance, Treg/T17 cell imbalance is also considered a crucial part of pathogenesis [9,10].T17, a particular subgroup of CD4+ T lymphocytes, can secrete high levels of cytokines such as IL-17A, a process modulated by TGF-β, IL-6, IL-21, IL-1, and IL-23, usually under the control of the transcription factor RORct. T17 cells can promote the maturation and diferentiation of neutrophils; with their strong chemotactic efect, T17 facilitates the infltration of neutrophils in the airway and contributes to the generation of the glucocorticoid-resistant phenotype of asthma.Moreover, T17 also plays a pivotal role in airway remodeling.Treg lymphocytes secrete anti-infammatory cytokines (IL-4, IL-10, TGF-β, etc.) to mediate immune tolerance (crucial in maintaining the body's immune balance) under the regulation of the transcription factor FoxP3.Both the defciency of Treg cells and the hyperactivity of T17 cells are together involved in the immunological pathogenesis of asthma, consistent with previous reports [9,18].
Although several infammatory cytokines were quantitated in this study, others such as IL-1β, TNF-α, and MCP-1 were not included due to a limited volume of samples.Numerous prior studies have shown that a non-type 2 immune response includes the release of several proinfammatory cytokines such as IL-1β, IL-6, IL-8, IL-17, and IFN-c [3,19].
Relevant research on asthma has shown that TP has a defnite anti-infammatory efect, but there are yet few reports of using aerosolized TP in animal models of asthma.In a rat model of asthma, nebulized TP can inhibit ASM hyperplasia by down-regulating NF-κB, bcl-2, and PI3K [20], but details of how to instil aerosolized TP into the airway were limited.Triptolide has a molecular formula of C 20 H 24 O 6 and a molecular weight of 360.4.It is insoluble in water, but soluble in chloroform, dichloromethane, and dimethyl sulfoxide (DMSO), indicating stronger lipid solubility.As a reference, budesonide has been commonly used in clinical practice as an inhaled glucocorticoid for nearly 30 years; its molecular formula is C 25 H 34 O 6 , its molecular weight is 450.3, and it is soluble in both water and dichloromethane.Another drug approved by the FDA for clinical atomization use is the antibiotic tobramycin (molecular formula: C 18 H 37 N 5 O 9 , molecular weight: 467.5), which is soluble in water but poorly soluble in 100% ethanol (molecular diagrams of the above are shown in Figure 6).Te three molecules are all alicyclic hydrocarbon structures with 3-5 rings.Te smaller molecular weight and stronger lipid solubility of TP may facilitate its entry into the airway epithelium with its anti-infammatory efects.
Te specifc binding target of TP is ERCC3 (also known as XBP), the core subunit of TFIIH.Meanwhile, the most related signaling pathway involved in the mechanisms of TP were the NF-κB and MAPK signaling pathways [13].According to a relatively early research, TP could suppress the dendritic cell-mediated chemoattraction of neutrophils and T cells by inhibiting Stat3 phosphorylation and NF-κB activation [13].Aldactone can induce the degradation of XBP, which consequently reduces the level of infammation in the cardiovascular system through regulating NF-κB and AP-1 signaling pathways [21].Hence, XBP might be a promising therapeutic target for some chronic infammatory diseases.Meanwhile, transcription of IL-17 mRNA dropped more in the Dex group than in the TP group (P � 0.0013).mRNA expression of FoxP3 in the NS group was signifcantly decreased compared with the two intervention groups (P ≤ 0.001), and FoxP3 transcription in the TP group was lower than in the Dex group (P � 0.0015).mRNA expression of RORct varied in this order: NS > TP > Dex (P ≤ 0.001).Canadian Respiratory Journal

Conclusion
Tis study revealed that the inhalation of aerosolized triptolide is efective at reducing infammation in a mouse model of asthma, though less than intraperitoneal injections of Dex.Tis is a proof-of-concept study which is an initial step for assessing a potential role for TP in asthma treatment.
Tere are still numerous steps to be clarifed and optimized, such as the exact molecular mechanism, ideal solvent, suitable dosage, and minimization of toxicity and side effects, all of which merit further investigation.Nebulized medicines have the advantages of direct and rapid action, convenient usage, relatively low doses, and few adverse reactions, so it will be interesting to extend atomized TP inhalation to preclinical studies.

Figure 1 :
Figure1: ELISA analysis of infammatory cytokines in BALF from mice sensitized with OVA and treated with normal saline (NS), the positive control anti-infammatory Dex, or the test compound TP.Compared with the other two intervention groups, IL-6 was increased signifcantly in the NS group, and it was decreased more in the Dex group than in the TP group (P ≤ 0.001).Te anti-infammatory cytokine IL-10 varied signifcantly in this order: NS < TP < Dex (P < 0.001), while IL-17, IL-23, and TGF-β showed variable results, with reduced levels in TP and Dex-treated mice (P ≤ 0.001).Te units are ng/ml for TGF-β1 and pg/ml for the other indexes.

Figure 2 :
Figure2: RT-PCR analysis of relevant transcription factors in lung tissue (according to ΔCt between the other TFs and endogenous reference GAPDH).Tere was signifcantly higher expression of IL-17 mRNA in the NS than in the other intervention groups (P < 0.001).Meanwhile, transcription of IL-17 mRNA dropped more in the Dex group than in the TP group (P � 0.0013).mRNA expression of FoxP3 in the NS group was signifcantly decreased compared with the two intervention groups (P ≤ 0.001), and FoxP3 transcription in the TP group was lower than in the Dex group (P � 0.0015).mRNA expression of RORct varied in this order: NS > TP > Dex (P ≤ 0.001).

Figure 5 :
Figure 5: Pathological features of lung tissue in diferent groups.(A) NS group, (B) Dex group, (C) TP group, and (D) no-treatment control.Te images shown are representative of sections from four mice.White blood cell infltration, thickened alveoli septum, and alveoli flled with secretions (including RBC coagulation in vessels, highlighted with arrow heads) are shown.(HE staining, ×100).

Table 2 :
Changes in relevant cytokines in BALF.

Table 3 :
Changes in mRNA transcription of three TFs and expression of CD17+ T lymphocytes.