Danggui Buxue Tang Ameliorates Bleomycin-Induced Pulmonary Fibrosis by Suppressing the TLR4/NLRP3 Signaling Pathway in Rats

Abstract

Objective: To investigate the effects of Danggui Buxue Tang (DBT) on rats with pulmonary fibrosis (PF) and the underlying mechanism.

Methods: Sixty specific pathogen-free (SPF) male Sprague-Dawley (SD) rats were randomly divided into 4 groups: control, PF, prednisone treatment, and DBT treatment. Intratracheal instillation of bleomycin (BLM) was performed to establish a PF rat model. DBT was administered to PF rats concurrently for 2 weeks. Lung samples were then collected for HE and Masson staining after pulmonary function testing, and semiquantitative analysis for the degree of alveolitis and fibrosis was performed using the Szapiel and Ashcroft score systems. Myeloperoxidase (MPO) activity, hydroxyproline (HYP), hyaluronic acid (HA), and inflammatory cytokine content were measured. Western blotting was performed to detect fibrotic marker and TLR4/NLRP3 signaling pathway changes.

Results: Oral administration of DBT attenuated weight loss, survival rate, and pulmonary index. Lung histopathologic lesions were also reduced. DBT inhibited PF by decreasing the secretion of inflammatory cytokines and collagen deposition. Specifically, DBT reduced tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), IL-6, HYP, alpha-smooth muscle actin (α-SMA), collagen I, and collagen III levels. Corollary experiments identified a potential mechanism involving suppression of TLR4/MyD88/NF-κB signaling pathway activation and the NLRP3/ASC/caspase-1 axis, the downstream regulatory pathway.

Conclusion: DBT exhibited a potent effect on BLM-induced PF rats by inhibiting the TLR4/NLRP3 signaling pathway. Thus, DBT alleviates pulmonary inflammation to inhibit fibrotic pathology and should be considered as a candidate for the clinical treatment of PF.

Figure 1

Effect of DBT on bodyweight, food intake, survival time, and pulmonary index in PF rats. (a) Bodyweight changes in 4 groups (n = 8–15). (b) Food intake changes in 4 groups (n = 6). (c) Percent survival in 4 groups (n = 15). (d) Pulmonary index changes in 4 groups (n = 8). ^∗P < 0.05 versus the sham group and ^#P < 0.05 versus the model group.

Figure 2

Effect of DBT on pulmonary function in rats with PF. Ers: elastance; H: tissue elastance; Rn : Newtonian airway resistance; Rrs: total respiratory resistance; Crs: compliance; G: tissue damping; n = 6 for each group. ^∗∗P < 0.01 versus sham group and ^##P < 0.01 versus model group.

Figure 3

Effect of DBT on pulmonary inflammation in rats with PF. (a) Pathologic changes of lung tissues evaluated by H&E staining (scale bar = 50 μm) and assessed by the Szapiel score (n = 18). (b) Cell counts and classification in BALF (n = 8). (c) MPO activity tested by an examination kit (n = 8). (d) Levels of TNF-α, IL-6, and IL-1β in lung tissues examined by ELISA (n = 8). ^∗P < 0.05 and ^∗∗P < 0.01 versus sham group; ^#P < 0.05 and ^##P < 0.01 versus model group.

Figure 4

Effect of DBT on fibrotic markers and collagen production in rats with PF. (a) Masson-trichrome staining of lung tissues (scale bar = 50 μm) and Ashcroft scores (n = 18). (b) HA and HYP levels in lung tissue (n = 8). (c) Alpha-SMA, collagen I, and collagen III proteins examined by Western blotting (n = 3). 1, sham group; 2, model group; 3, prednisone group; 4, DBT group. ^∗∗P < 0.01 versus sham group; ^#P < 0.05 and ^##P < 0.01 versus model group.

Figure 5

Effect of DBT on the TLR4/MyD88/NF-κB signaling pathway. Western blotting was used for analysis. 1, sham group; 2, model group; 3, prednisone group; and 4, DBT group. ^∗P < 0.05 and ^∗∗P < 0.01 versus sham group; ^#P < 0.05 and ^##P < 0.01 versus model group; n = 3 in each group.

Figure 6

Effect of DBT on the NLRP3/ASC/caspase 1 signaling pathway. Western blotting was used for analysis. 1, sham group; 2, model group; 3, prednisone group; and 4, DBT group. ^∗∗P < 0.01 versus sham group; ^#P < 0.05 and ^##P < 0.01 versus model group; n = 3 in each group.