Pulmonary fibrosis (PF) is a chronic, progressive, and devastating interstitial lung disease mainly resulting from toxic insults, autoimmune injuries, drug-induced injuries, infectious injuries, or traumatic injuries. As can be affected by age, genetic predisposing factors, and environmental exposures, PF produces many different pathological types, which are mainly classified as idiopathic pulmonary fibrosis (IPF) and fibrosing alveolitis. PF is mainly characterized by diffuse pneumonia in the early period, secreting pro-inflammatory cytokines such as the tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6. Alveolar epithelial cell (AEC) injury and excessive deposition of collagen in extracellular matrix (ECM) ultimately result in progressive scarring and loss of lung function (Wynn and Vannella, 2016). With approximately 3 million people being affected worldwide, as shown in the related literature, IPF has an increasing burden (George et al., 2020). At present, there is no effective means or drugs that can ameliorate PF except lung transplantation in patients with a median survival of 3–5 years from the time of diagnosis (Meyer, 2017).‬‬‬‬‬‬‬‬‬‬‬‬

PF, with clinical manifestations such as progressive dyspnea, cough, sputum, chest pain, vomiting, dry mouth, dry throat, shortness of breath, and fatigue, was defined as “lung impediment” or “lung wilting” in books of traditional Chinese medicine, and its syndrome types were divided into lung–kidney Qi deficiency (Fei shen qi xu), dual deficiency of the lung and spleen (Pi fei liang xu), Qi deficiency with blood stasis (Qi xu xue yu), and phlegm obstructing the vessels and collaterals (Tan zu mai luo). They were mainly treated by methods of invigorating the lungs and kidneys (Bu fei yi shen), fortifying the spleen and boosting the lung (Jian pi yi fei), promoting blood circulation and dredging collaterals (Huo xue tong luo), transforming dampness and dispelling phlegm (Hua shi qu tan), and harmonizing and percolating dampness (He jie shen shi). The treatment process used decoctions including Schisandra decoction, Bufei decoction, and Buyang Huanwu decoction , and heat-clearing and toxin-resolving Chinese medicinal materials such as Centella asiatica (L.) Urban, Ophiopogon japonicus (L.f.) Ker-Gawl., and Houttuynia cordata Thunb. (family: Saururaceae) (H. cordata). The traditional Chinese medicine (TCM) mainly produces synergistic effects via the synergistic mechanism of actions with the active ingredients, enhancing functions, and producing less toxicity, which makes enormous contributions to modern drug development and disease treatment. Some active ingredients in the traditional Chinese medicine play a good role in the progression of PF, just like paclitaxel (PTX) (Wang et al., 2013), tanshinone IIA (Tan IIA) (He et al., 2015), and andrographolide (Andro) (Guo et al., 2016).

H. cordata, famous in China, Japan, Korea, and Northeast India for its medicinal properties, is used in the treatment of a number of diseases such as pneumonia, diabetes, and even SARS (Lau et al., 2008). Studies have found that H. cordata also has a certain inhibitory effect on PF; for instance, the volatile oil of H. cordata contains 4-terpineol, α-terpineol, l-bornyl acetate, and methyl-n-nonyl ketone, and has good anti-inflammatory and anti-pulmonary fibrosis effects in vitro and in vivo (Du et al., 2012). The aqueous extract of H. cordata has effects of anti-pulmonary fibrosis by inhibiting the expression of superoxide dismutase, malondialdehyde, hydroxyproline, interferon-γ (IFN-γ), and TNF-α (Ng et al., 2007). Qing fei xie ding granules (Sun et al., 2018), mainly including Ephedra sinica, apricot kernels, and H. cordata, have a protective effect on PF as a novel Chinese traditional patent medicine. However, the anti-pulmonary fibrosis mode and the mechanism of action of H. cordata are not fully clarified. To address the protective effects of H. cordata on PF, our previous study has shown that sodium houttuyfonate is an attractive candidate alleviating IPF and pulmonary toxicity induced by BLM through downregulation of TGFβ1-Smad2/3 signal transduction (Shen et al., 2021).

Despite the obvious medicinal effects and clinical uses of TCM, little has been elucidated regarding the underlying molecular mechanisms. Network pharmacology integrates several disciplines, including systems biology, network biology, computational biology, multi-target pharmacology, and molecular pharmacology. And the core is the construction of a drug molecule–target–disease link network and the systematic analysis of the three relationships. It can be used to predict active molecules and actions of TCM, and then to search for new pharmacological mechanisms of action and broaden the indications of TCM, etc. Molecular docking is used to design drugs by in silico modeling of the interaction of target proteins with drug molecules. Molecular docking has been gradually applied to reveal the relevant mechanism of action of drugs in the development of new drugs. This study aims to utilize network pharmacology and molecular docking to seek the active compounds of H. cordata, construct the drug–compounds–genes–disease network, and to analyze the underlying mechanism of H. cordata in the treatment of PF.

This study reveals the mechanism of the anti-pulmonary fibrosis pharmacological action of H. cordata by a TCM network pharmacology–based strategy and provides ideas for H. cordata for further drug development. This workflow is shown in Figure 1.

PF is a progressive and severe condition characterized by mesenchymal cell proliferation and differentiation, ECM deposition, and remodeling of the lung parenchyma and airways. Although progress has been made in the research on PF, our understanding of its pathological mechanism remains rudimentary. There is still no complete cure for PF, and current therapies merely act to delay but not completely stop the progression of the disease.

H. cordata, a Chinese herb with special odor, is mainly used to treat bronchitis and upper respiratory tract infection. And studies have reported that it can inhibit rapid PF by upregulating IFN-γ and inhibiting the TGF-β1/Smad pathway (Du et al., 2012). 2-Undecanone, houttuynin, and quercetin are the main constituents of H. cordata. Houttuynin is the easily polymerized major constituent of the volatile oil, and the content is usually about 15%, which was first isolated from H. cordata in 1952 (Pan et al., 2010). Because houttuynin was unstable in chemical characteristics, sodium new houttuyfonate (sodium lauroyl-α-hydroxyethyl sulfonate, C₁₄H₂₇NaO₅S, MW = 330.4), an adduct compound of houttuynin and sodium bisulfite, was developed and put in use. Sodium new houttuyfonate has better water solubility and stability and can be transformed into houttuynin under physiological conditions. Previous studies have shown that the lung is the target organ of houttuynin, and it has good therapeutic effects on pneumonia (Deng et al., 2012). Also, sodium new houttuyfonate is an effective clinical therapeutic agent applied to respiratory infection and inflammation such as acute or chronic bronchitis and pneumonia. 2-Undecanone is used as a standard marker for the quality control of H. cordata in the Chinese pharmacopoeia, and its content is usually about 17.0–27.5% (Yu-hong et al., 2015). Studies have shown that 2-undecanone performed various types of pharmacological functions, including anti-inflammatory and antitumor effects (Chen et al., 2014; Lou et al., 2019). Quercetin is one of the major flavonoid-active components in H. cordata, and the content is usually about 0.50 mg/g (Xiao-lei and Xiao-yin, 2013), which could overexpress the FasL receptor and caveolin-1 expression, reducing AKT activation and eliminating apoptosis resistance in IPF (Hohmann et al., 2019). Quercetin increases the Nrf2 activity to defend against oxidation, restore the disturbed redox balance, and reduce inflammation (Veith et al., 2017). As has been reported, quercetin can also reduce the expression of IL-6, TNF-α, and IL-1β in the bronchoalveolar lavage fluid of bleomycin-induced PF in vivo (Baowen et al., 2010).

Our results analyzed the ingredients in H. cordata, as well as the functions and potential targets against PF. According to the topological property analysis of the “drug–disease” network and the results of the PPI network analysis, the targets of the action of H. cordata were members of the mitogen-activated protein kinase (MAPK) family, TNF, AKT, or the cytokine VEGF. The results of the GO functional enrichment analysis revealed that H. cordata is mainly involved in signal transduction, inflammatory immunity, regulation of chemokine production, etc. The KEGG enrichment analysis showed that the possible pathways involved in the anti-pulmonary fibrosis of H. cordata mainly included the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway, MAPK signaling pathway, TNF signaling pathway, IL-17 signaling pathway, HIF-1 signaling pathway, and NOD-like receptor signaling pathway. All the above signaling pathways are related to inflammation and immune responses, indicating that H. cordata exerts immunomodulatory and anti-inflammatory effects through multiple pathways, exactly in accordance with our previous study which shows that sodium houttuyfonate reduces BLM-induced elevation of inflammatory cytokines such as IL-1, IL-6, and TNF-α in mice (Shen et al., 2021). The PI3K/Akt signaling pathway mainly regulates cell proliferation, inflammation, and survival, and maintains the biological properties of malignant cells. PI3K, consisting of a regulatory subunit, p85, and a catalytic subunit, p110, primarily exist in the cytoplasm. Akt, also known as the protein kinase B (PKB), regulates and controls cell proliferation and apoptosis. After PI3K activation by tyrosine kinases, PIP3 was generated on the plasma membrane to interact with the PH domain of Akt, and Thr³⁰⁸ and Ser⁴⁷³ of Akt will be phosphorylated with the help of 3-phosphoinositide–dependent protein kinase 1 (PDK1) and PDK2, Akt-activated (Assinder et al., 2009). PI3K/Akt contributes to the development and progression of PF by upregulating cell growth and collagen expression (Lu et al., 2010). TNF-α, a key regulator of inflammation, could initiate and drive many pulmonary pathological diseases by inducing the accumulation of inflammatory cells, causing oxidative and nitrosative stress, airway hyperactivity, and tissue remodeling (Blaser et al., 2016). TNF-α stimulates epithelial cell proliferation and contributes to epithelial thickening and PF (Sasaki et al., 2000). MAPKs, serine–threonine protein kinases, have been proven to control cellular processes associated with fibrosis, such as myofibroblast transformation and others (Xu et al., 2004). The extracellular signal–regulated kinases 1 and 2 (ERK1/2) cascade can regulate cell proliferation, differentiation, EMT, and tumorigenesis. The activation of ERK signaling causes morphological changes and the downregulation of E-cadherin expression in epithelial cells (Grände et al., 2002). Next, molecular docking studies for MAPK1, AKT1, and TNF-α were performed using the active constituents 2-undecanone, quercitrin, β-stigmasterol, myrcene, isoquercitrin, heriguard, rutin, quercetin, and hyperoside to evaluate the thermodynamic binding effects of proteins and ligands. The results showed that the target proteins and components had good binding effects. And quercetin showed great binding strength with MAPK1, AKT1, and TNF-α, which may play an essential role in PF.

LPS, an endotoxin component of Gram-negative bacilli as an inflammatory inducer, is a vital promoter of acute lung injury (ALI) as well as promoting EMT formation in various cell types including alveolar epithelial cells (AECs) (Dong et al., 2017; Xiao et al., 2020). A549 cells were added to LPS coculture to induce EMT validating the predicted outcome of network pharmacology. Evidence showed that quercetin was well validated by its docking to IL-6, TNF, and IL-1β proteins (Yu et al., 2021). These findings strikingly supported the molecular docking and experimental results of our study that quercetin may exert anti-PF effects by targeted binding to TNF. And sodium houttuyfonate inhibited the expression of inflammatory cytokine TNF-α, which was consistent with our previous findings in bleomycin-induced pulmonary fibrosis in mice. Results from our study revealed the potential mechanism of H. cordata and suggested that H. cordata has a clinical application value in attenuating PF because of its effect in inhibiting the expression of AKT, p-AKT, p44/42 MAPK (ERK1/2), p-p44/42 MAPK (ERK1/2), p-p38 MAPK, and TNF-α via the PI3K/AKT, MAPK, and TNF signaling pathways. However, the mechanism needs more investigation in vitro and in vivo models.

Our present study has important implications for the current understanding of the molecular mechanism of H. cordata in anti-pulmonary fibrosis, which involves multicomponents, multi-targets, and multi-pathways. An in-depth study should be considered by taking H. cordata as a novel anti-pulmonary fibrotic drug.