Lian Hua Qing Wen Capsules, a Potent Epithelial Protector in Acute Lung Injury Model, Block Proapoptotic Communication Between Macrophages, and Alveolar Epithelial Cells

Abstract

Besides pathogen evading, Acute Lung Injury (ALI), featuring the systematic inflammation and severe epithelial damages, is widely believed to be the central non-infectious factor controlling the progression of infectious diseases. ALI is partly caused by host immune responses. Under the inspiration of unsuccessful treatment in COVID-19, recent insights into pathogen-host interactions are leading to identification and development of a wide range of host-directed therapies with different mechanisms of action. The interaction unit consisting of macrophages and the alveolar epithelial cells has recently revealed as the therapeutic basis targeting ALI. Lian Hua Qing Wen capsule is the most effective and commonly-used clinical formula in treating respiratory infection for thousands of years in China. However, little is known about its relevance with ALI, especially its protective role against ALI-induced alveolar tissue damages. Aiming to evaluate its contribution in antibiotics-integrating therapies, this study pharmacologically verified whether LHQW could alleviate lipopolysaccharide (LPS)-induced ALI and explore its potential mechanisms in maintaining the physiology of macrophage-epithelial unit. In ALI mouse model, the pathological parameters, including the anal temperature, inflammation condition, lung edema, histopathological structures, have all been systematically analyzed. Results consistently supported the effectiveness of the combined strategy for LHQW and low-dose antibiotics. Furthermore, we established the macrophages-alveolar epithelial cells co-culture model and firstly proved that LHQW inhibited LPS-induced ER stress and TRAIL secretion in macrophages, thereby efficiently protected epithelial cells against TRAIL-induced apoptosis. Mechanistically, results showed that LHQW significantly deactivated NF-κB and reversed the SOCS3 expression in inflammatory macrophages. Furthermore, we proved that the therapeutic effects of LHQW were highly dependent on JNK-AP1 regulation. In conclusion, our data proved that LHQW is an epithelial protector in ALI, implying its promising potential in antibiotic alternative therapy.

Keywords: Lian Hua Qing Wen capsule; acute lung injury; endoplasmic reticulum stress; macrophages and epithelial cells; tumor necrosis factor-related apoptosis-inducing ligand.

Figure 1

Lian Hua Qing Wen capsule (LHQW) significantly protected lung tissue from inflammatory damage and functioned synergistically with antibiotics in an lipopolysaccharide (LPS)-induced Acute Lung Injury (ALI) model. At 24 h after the last drug treatment, all mice were anesthetized with 4% chloral hydrate (0.4 g.kg^-1) and placed in the supine position on the surgical board. Control mice were instilled with saline, and others were treated with LPS (5 mg·kg-1). All of the above processes were performed on a ventilator. (A) Effects of LHQW on anal temperature. (B) Effects of LHQW on lung wet/dry ratio. (C) Effect of LHQW on MCP1 secretion. (D–F) Effects of LHQW on the measurement of infiltrated cells in BALF were measured with CCK-8 and microscope assays(40×). Lung tissue from ALI model mice were fixed in 4% PFA, then 3-5-μm sections were prepared after paraffin embedding. All sections were subjected to immunohistochemistry. (G, H) Effects of LHQW on the degree of edema by measuring Na, K ATPase levels in lung tissue (200×). The data was presented as the mean ± S.E.M.; n = 8 mice per group; *P < 0.05, **P < 0.01 vs. the LPS group.

Figure 2

Lian Hua Qing Wen capsule (LHQW) inhibited the expression and secretion of inflammatory molecules in macrophages. Mice were dissected 24 h later, the lungs were lavaged with saline, and BALF was collected. (A) Dual luciferase reporter analysis in THP1cells influenced by LHQW. (B, C) Detection of NF-κB pathway-related proteins phospho-p65, total-p65, phosphorylation-IKK, total-IKK, and IKB-α in THP1 cells. (D, E) Determination of adhesion molecules VCAM1 and ICAM1 by western blot assay. (F) LHQW decreased the secretion of TNF-α, IL-1B, and IL-6 measured by ELISA. (G, H) The expression of VCAM1, ICAM1, phospho-p65, and total-p65 in alveolar macrophages measured with western blot analysis. The data was presented as the mean ± S.E.M. of three independent experiments. ^# P < 0.05 vs. the NC group, ^* P < 0.05, ^** P < 0.01 vs. the LPS group. LHQW-L: 50 μg/ml; LHQW-M: 100 μg/ml; LHQW-H: 500 μg/ml.

Figure 3

Lian Hua Qing Wen capsule (LHQW) alleviated lipopolysaccharide (LPS)-induced endoplasmic reticulum stress (ERS) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in macrophages. Bronchoalveolar lavage fluid (BALF) samples were centrifuged, and pulmonary macrophages were isolated with the adherent method. THP1 cells were first induced by PMA to differentiate into macrophages, and subsequently treated with LHQW and LPS for another 24 h. (Togno-Peirce et al., 2013) In the cells, (A) LHQW inhibited the transcription of CHOP and TRAIL in THP1 cells detected by RT-PCR assays. (B) LHQW downregulated the expression of TRAIL secretion in the supernatant of THP1 cells detected by ELISA assay. (C) Treatment with the ERS inhibitor PBA reduced TRAIL concentration in the supernatant of THP1 cells. (D, E) LHQW inhibited the translation of CHOP and GRP78 in THP1 cells and RAW 264.7 cells detected by western blot. (F) Effect of LHQW treatment on the expression of DR5, the ligand of TRAIL, in HPA cells treated with the supernatant from THP1 cells. (G, H) The translation of CHOP in pulmonary macrophages. (I) The secretion of TRAIL in BALF. The data weas presented as the mean ± S.E.M. of three independent experiments. ^# P < 0.05 vs. the NC group. ^* P < 0.05, ^** P < 0.01 vs. the LPS group. LHQW-L: 50 μg/mL; LHQW-M: 100 μg/mL; LHQW-H: 500 μg/mL. GRP78.

Figure 4

Lian Hua Qing Wen capsule (LHQW) protected alveolar epithelial cells against TRAIL-induced apoptosis. Lung tissue come from ALI model mice were fixed in 4% PFA, then 3-5-μm sections were prepared after paraffin embedding. All sections were subjected to immunohistochemistry. In the macrophage-epithelial co-cultural model, human pulmonary alveolar epithelial cells (HPA cells) in 6-well plates were incubated with supernatant from THP1 cells that were treated with LHQW, tunicamycin (TM), and lipopolysaccharide (LPS). (A) The effect of LHQW on increasing the cell counting of HPA cells in the macrophage-epithelial co-cultural model, which was based on MTT assays. (B, C) LHQW alleviated apoptosis in the macrophage-epithelial co-cultural model measured by (B) AV-PI assay and (C) caspase3 activity. (D) The number of apoptotic cells (dark brown) in lung tissue measured by TUNEL (200×). THP1 cells in 6-well plates were first treated with PMA for 24 h, followed by LHQW and LPS for another 24 h, then the supernatant was collected. The data was presented as the mean ± S.E.M. of three independent experiments. ^# P < 0.05 vs. the NC group. ^* P < 0.05, ^** P < 0.01 vs. the LPS group. LHQW-L: 50 μg/ml; LHQW-M: 100 μg/ml; LHQW-H: 500 μg/ml; TM: tunicamycin.

Figure 5

Lian Hua Qing Wen capsule (LHQW) specifically inhibited endoplasmic reticulum stress (ERS) through c-Jun N-terminal kinase (JNK) pathway activation. Bronchoalveolar lavage fluid (BALF) samples were centrifuged, and pulmonary macrophages were isolated with the adherent method. THP1 cells were first induced by PMA to differentiate into macrophages, and subsequently treated with LHQW and LPS for another 24 h. RAW 264.7 cells were also treated with LHQW and LPS for 24 h. (Togno-Peirce et al., 2013). In the cells, (A, B) LHQW enhanced SOCS3 expression in (A) THP1 cells, (B) RAW 264.7 cells by utilizing western blot. (C) LHQW decreased phospho-JNK expression by western blot analysis. In the tissues, (D) LHQW enhanced the translation of SOCS3 in alveolar macrophages. The data was presented as the mean ± S.E.M. of three independent experiments. ^# P < 0.05 vs. the NC group. ^* P < 0.05, ^** P < 0.01 vs. the LPS group. LHQW-L: 50 μg/ml; LHQW-M: 100 μg/ml; LHQW-H: 500 μg/ml; TM, tunicamycin.