COVID-19: Immunohistochemical Analysis of TGF-β Signaling Pathways in Pulmonary Fibrosis

Acute respiratory distress syndrome (ARDS) followed by repair with lung remodeling is observed in COVID-19. These findings can lead to pulmonary terminal fibrosis, a form of irreversible sequelae. There is evidence that TGF-β is intimately involved in the fibrogenic process. When activated, TGF-β promotes the differentiation of fibroblasts into myofibroblasts and regulates the remodeling of the extracellular matrix (ECM). In this sense, the present study evaluated the histopathological features and immunohistochemical biomarkers (ACE-2, AKT-1, Caveolin-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 tissue expression) involved in the TGF-β1 signaling pathways and pulmonary fibrosis. The study consisted of 24 paraffin lung samples from patients who died of COVID-19 (COVID-19 group), compared to 10 lung samples from patients who died of H1N1pdm09 (H1N1 group) and 11 lung samples from patients who died of different causes, with no lung injury (CONTROL group). In addition to the presence of alveolar septal fibrosis, diffuse alveolar damage (DAD) was found to be significantly increased in the COVID-19 group, associated with a higher density of Collagen I (mature) and III (immature). There was also a significant increase observed in the immunoexpression of tissue biomarkers ACE-2, AKT-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 in the COVID-19 group. A significantly lower expression of Caveolin-1 was also found in this group. The results suggest the participation of TGF-β pathways in the development process of pulmonary fibrosis. Thus, it would be plausible to consider therapy with TGF-β inhibitors in those patients recovered from COVID-19 to mitigate a possible development of pulmonary fibrosis and its consequences for post-COVID-19 life quality.

Download Full-text

Potential Mechanism Prediction of Herbal Medicine for Pulmonary Fibrosis Associated with SARS-CoV-2 Infection Based on Network Analysis and Molecular Docking

Frontiers in Pharmacology ◽

10.3389/fphar.2021.602218 ◽

2021 ◽

Vol 12 ◽

Author(s):

De Jin ◽

Xuedong An ◽

Yuqing Zhang ◽

Shenghui Zhao ◽

Liyun Duan ◽

...

Keyword(s):

Molecular Docking ◽

Pulmonary Fibrosis ◽

Signaling Pathways ◽

Topological Analysis ◽

Recovery Period ◽

Treatment Protocol ◽

Docking Simulation ◽

Mapk Signaling ◽

Tnf Α ◽

Tgf Β1

Background: Coronavirus Disease 2019 (COVID-19) is still a relevant global problem. Although some patients have recovered from COVID-19, the sequalae to the SARS-CoV-2 infection may include pulmonary fibrosis, which may contribute to considerable economic burden and health-care challenges. Convalescent Chinese Prescription (CCP) has been widely used during the COVID-19 recovery period for patients who were at high risk of pulmonary fibrosis and is recommended by the Diagnosis and Treatment Protocol for COVID-19 (Trial Version sixth, seventh). However, its underlying mechanism is still unclear.Methods: In this study, an integrated pharmacology approach was implemented, which involved evaluation of absorption, distribution, metabolism and excretion of CCP, data mining of the disease targets, protein-protein interaction (PPI) network construction, and analysis, enrichment analysis, and molecular docking simulation, to predict the bioactive components, potential targets, and molecular mechanism of CCP for pulmonary fibrosis associated with SARS-CoV-2 infection.Results: The active compound of CCP and the candidate targets, including pulmonary fibrosis targets, were obtained through database mining. The Drug-Disease network was constructed. Sixty-five key targets were identified by topological analysis. The findings of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation suggested that the VEGF, Toll-like 4 receptor, MAPK signaling pathway, and TGF-β1 signaling pathways may be involved in pulmonary fibrosis. In the molecular docking analyses, VEGF, TNF-α, IL-6, MMP9 exhibited good binding activity. Findings from our study indicated that CCP could inhibit the expression of VEGF, TNF-α, IL-6, MMP9, TGF-β1 via the VEGF, Toll-like 4 receptor, MAPK, and TGF-β1 signaling pathways.Conclusion: Potential mechanisms involved in CCP treatment for COVID-19 pulmonary fibrosis associated with SARS-CoV-2 infection involves multiple components and multiple target points as well as multiple pathways. These findings may offer a profile for further investigations of the anti-fibrotic mechanism of CCP.

Download Full-text

Astragaloside IV Synergizing with Ferulic Acid Ameliorates Pulmonary Fibrosis by TGF-β1/Smad3 Signaling

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/8845798 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Jiahuan Tong ◽

Zhisong Wu ◽

Yuchen Wang ◽

Qingxun Hao ◽

Haoge Liu ◽

...

Keyword(s):

Oxidative Stress ◽

Pulmonary Fibrosis ◽

Ferulic Acid ◽

Lung Tissue ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Sham Operation ◽

Astragaloside Iv ◽

Group A ◽

Tgf Β1

Objective. The study aims to research the interventional effect and mechanism of astragaloside IV (Ast) synergizing with ferulic acid (FA) on idiopathic pulmonary fibrosis (IPF) induced by bleomycin in mice. Methods. The mice were randomly divided into seven groups with 10 mice in each group, namely, a sham operation group, a model group, a miRNA-29b (miR-29) group, a miR-29b negative control group (NC group), a FA group, an Ast group, and a combination group. A mouse model of pulmonary fibrosis was established by intratracheal instillation of bleomycin. Samples were collected after 28 days of continuous administration. Hematoxylin and eosin (HE) and Masson staining were used to observe pathological changes in the lung tissue, and the degree of fibrosis was evaluated using the hydroxyproline content. Changes in transforming growth factor-β1 (TGF-β1) and Smad3 in the lung were observed using immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) in the serum. PCR was used to detect the expression of the miR-29b, TGF-β1, Smad3, and nuclear factor E2-related factor 2 (Nrf2) genes. Western blotting was used to detect the content of the TGF-β/Smad3 protein. Results. Ferulic acid combined with astragaloside IV reduced the degree of pulmonary fibrosis and the synthesis of hydroxyproline in lung tissue. The combination of the two also regulated the oxidative stress response ， TGF-β1/Smad3 pathway and miR-29b in lung tissue. Conclusion. Astragaloside IV combined with ferulic acid regulated the oxidative stress of lung tissues and TGF-β1/Smad3 signaling through miR-29b, thereby reducing the degree of pulmonary fibrosis. This provides a reference direction for the clinical treatment of IPF patients.

Download Full-text

Amelioration of paraquat-induced pulmonary fibrosis in mice by regulating miR-140-5p expression with the fibrogenic inhibitor Xuebijing

International Journal of Immunopathology and Pharmacology ◽

10.1177/2058738420923911 ◽

2020 ◽

Vol 34 ◽

pp. 205873842092391 ◽

Cited By ~ 1

Author(s):

Min-na Dong ◽

Yun Xiao ◽

Yun-fei Li ◽

Dong-mei Wang ◽

Ya-ping Qu ◽

...

Keyword(s):

Growth Factor ◽

Treatment Group ◽

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Lavage Fluid ◽

Tissue Growth ◽

Control Treatment ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Tgf Β1

Intravenous Xuebijing (XBJ) therapy suppresses paraquat (PQ)-induced pulmonary fibrosis. However, the mechanism underlying this suppression remains unknown. This work aimed to analyze the miR-140-5p-induced effects of XBJ injection on PQ-induced pulmonary fibrosis in mice. The mice were arbitrarily assigned to four groups. The model group was administered with PQ only. The PQ treatment group was administered with PQ and XBJ. The control group was administered with saline only. The control treatment group was administered with XBJ only. The miR-140-5p and miR-140-5p knockout animal models were overexpressed. The gene expression levels of miR-140-5p, transglutaminase-2 (TG2), β-catenin, Wnt-1, connective tissue growth factor (CTGF), mothers against decapentaplegic homolog (Smad), and transforming growth factor-β1 (TGF-β1) in the lungs were assayed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. The levels of TGF-β1, CTGF, and matrix metalloproteinase-9 (MMP-9) in the bronchoalveolar lavage fluid were assessed by enzyme-linked immunosorbent assay (ELISA). Hydroxyproline (Hyp) levels and pulmonary fibrosis were also scored. After 14 days of PQ induction of pulmonary fibrosis, AdCMV-miR-140-5p, and XBJ upregulated miR-140-5p expression; blocked the expressions of TG2, Wnt-1, and β-catenin; and decreased p-Smad2, p-Smad3, CTGF, MMP-9, and TGF-β1 expressions. In addition, Hyp and pulmonary fibrosis scores in XBJ-treated mice decreased. Histological results confirmed that PQ-induced pulmonary fibrosis in XBJ-treated lungs was attenuated. TG2 expression and the Wnt-1/β-catenin signaling pathway were suppressed by the elevated levels of miR-140-5p expression. This inhibition was pivotal in the protective effect of XBJ against PQ-induced pulmonary fibrosis. Thus, XBJ efficiently alleviated PQ-induced pulmonary fibrosis in mice.

Download Full-text

Role of the Hippo signaling pathway in safflower yellow pigment treatment of paraquat-induced pulmonary fibrosis

Journal of International Medical Research ◽

10.1177/0300060520905425 ◽

2020 ◽

Vol 48 (9) ◽

pp. 030006052090542

Author(s):

Hai Li ◽

Baotian Kan ◽

Lingli Song ◽

Yufa Liu ◽

Xiangdong Jian

Keyword(s):

Growth Factor ◽

Pulmonary Fibrosis ◽

Quantitative Polymerase Chain Reaction ◽

Tissue Growth ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Therapeutic Effects ◽

Hippo Signaling ◽

Exposure Group ◽

Tgf Β1

Objective To elucidate the molecular mechanisms by which safflower yellow (SY) mediates therapeutic effects in rats with paraquat intoxication-induced pulmonary fibrosis. Methods Rats received combinations of paraquat, SY, and SB431542, a transforming growth factor (TGF)-β1 receptor antagonist. Survival over 28 days was assessed by Kaplan–Meier analysis. Rat tissue and serum samples were assessed by hematoxylin and eosin staining, Masson’s Trichrome staining, immunoblotting, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and transmission electron microscopy. Results Survival rates were higher in SY and SB431542 groups (treatment and paraquat) than in the exposure group (paraquat alone). In the exposure group, serum TGF-β1 levels increased between days 3 and 14; mammalian STE20-like (MST) levels increased between days 3 and 7; TGF-β1 and Smad3 levels increased between days 3 and 14; and Yap and connective tissue growth factor levels increased between days 3 and 28. TGF-β1 levels were lower in SY and SB431542 groups than in the exposure group. Pathology scores were higher in exposure, SY, and SB431542 groups than in the control group throughout the experiment. Conclusions In rats with paraquat intoxication-induced pulmonary fibrosis, Hippo signaling could be activated by the MST-Yap pathway; SY and SB431542 could alleviate pulmonary fibrosis via Hippo signaling.

Download Full-text

Losartan attenuates paraquat-induced pulmonary fibrosis in rats

Human & Experimental Toxicology ◽

10.1177/0960327114543840 ◽

2014 ◽

Vol 34 (5) ◽

pp. 497-505 ◽

Cited By ~ 11

Author(s):

F Guo ◽

YB Sun ◽

L Su ◽

S Li ◽

ZF Liu ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Lung Injury ◽

Mrna Expression ◽

Collagen Type ◽

Collagen Type I ◽

Control Group ◽

Type I ◽

Expression Levels ◽

Relative Expression ◽

Tgf Β1

Paraquat (PQ) is one of the most widely used herbicides in the world and can cause pulmonary fibrosis in the cases with intoxication. Losartan, an angiotensin II type 1 receptor antagonist, has beneficial effects on the treatment of fibrosis. The aim of this study was to examine the effect of losartan on pulmonary fibrosis in PQ-intoxicated rats. Adult male Sprague Dawley rats ( n = 32, 180–220 g) were randomly assigned to four groups: (i) control group; (ii) PQ group; (iii) PQ + losartan 7d group; and (iv) PQ + losartan 14d group. Losartan treatment (intragastrically (i.g.), 10 mg/kg) was performed for 7 and 14 days after a single i.g. dose of 40 mg/kg PQ. All rats were killed on the 16th day, and hematoxylin–eosin and Masson’s trichrome staining were used to examine lung injury and fibrosis. The levels of hydroxyproline and transforming growth factor β1 (TGF-β1), matrix metallopeptidase 9 (Mmp9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) messenger RNA (mRNA) expression and relative expression levels of collagen type I and III were also detected. PQ caused a significant increase in hydroxyproline content, mRNA expression of TGF-β1, Mmp9, and TIMP-1, and relative expression levels of collagen type I and III ( p < 0.05), while losartan significantly decreased the amount of hydroxyproline and downregulated TGF-β1, Mmp9, and TIMP-1 mRNA and collagen type I and III expressions ( p < 0.05). Histological examination of PQ-treated rats showed lung injury and widespread inflammatory cell infiltration in the alveolar space and pulmonary fibrosis, while losartan could markedly reduce such damage and prevent pulmonary fibrosis. The results of this study indicated that losartan could reduce lung damage and prevent pulmonary fibrosis induced by PQ.

Download Full-text

IL-4/IL-13 Remodeling Pathway of Covid-19 Lung Injury

10.21203/rs.3.rs-34688/v2 ◽

2020 ◽

Author(s):

Caroline Busatta Vaz de Paula ◽

Marina Luise Viola Azevedo ◽

Seigo Nagashima ◽

Ana Paula Camargo Martins ◽

Mineia Alessandra Scaranello Malaquias ◽

...

Keyword(s):

Lung Injury ◽

Transforming Growth Factor Beta ◽

Influenza A ◽

Transforming Growth Factor ◽

Diffuse Alveolar Damage ◽

Tissue Expression ◽

Lung Damage ◽

Control Group ◽

M2 Macrophages ◽

Th17 Response

Abstract Background: The COVID-19 fatality rate is high when compared to the H1N1pdm09 (pandemic Influenza A virus H1N1 subtype) rate, and although both cause an aggravated inflammatory response, the differences in the mechanisms of both pandemic pneumonias need clarification.Objective: To analyze tissue expression of interleukins 4, 13, (IL-4, IL-13), transforming growth factor-beta (TGF-β), and the number of M2 macrophages (Sphingosine-1) in patients who died by COVID-19, comparing with cases of severe pneumopathy caused by H1N1pdm09, and a control group without lung injury.Methods: Six lung biopsy samples of patients who died of SARS-CoV-2 (COVID-19 group) were used and compared with ten lung samples of adults who died from a severe infection of H1N1pdm09 (H1N1 group) and eleven samples of patients who died from different causes without lung injury (CONTROL group). The expression of IL-4, IL-13, TGF-β, and M2 macrophages score (Sphingosine-1) were identified through immunohistochemistry (IHC).Results and conclusion: Significantly higher IL-4 tissue expression and Sphingosine-1 in M2 macrophages was observed in the COVID-19 group when compared to both the H1N1 and the CONTROL groups. Different mechanism of diffuse alveolar damage (DAD) in SARS-CoV-2 compared to H1N1pdm09 infections were observed. IL-4 expression and lung remodeling are phenomena observed in both SARS-COV-2 and H1N1pdm09. However, SARS-CoV-2 seems to promote lung damage through different mechanisms, such as the scarce participation Th1/Th17 response and the higher participation of the Th2. The understanding and management of the aggravated and ineffective immune response elicited by SARS-CoV-2 merits further clarification to improve treatments propose.

Download Full-text

Catalpol Protects Against Pulmonary Fibrosis Through Inhibiting TGF-β1/Smad3 and Wnt/β-Catenin Signaling Pathways

Frontiers in Pharmacology ◽

10.3389/fphar.2020.594139 ◽

2021 ◽

Vol 11 ◽

Author(s):

Fan Yang ◽

Zhen-feng Hou ◽

Hao-yue Zhu ◽

Xiao-xuan Chen ◽

Wan-yang Li ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Signaling Pathways ◽

Molecular Mechanisms ◽

Epithelial Mesenchymal Transition ◽

Gene Expression Omnibus ◽

Matrix Remodeling ◽

Mesenchymal Transition ◽

Collagen Remodeling ◽

Gsk 3Β ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by fibroblast proliferation and extracellular matrix remodeling; however, the molecular mechanisms underlying its occurrence and development are not yet fully understood. Despite it having a variety of beneficial pharmacological activities, the effects of catalpol (CAT), which is extracted from Rehmannia glutinosa, in IPF are not known. In this study, the differentially expressed genes, proteins, and pathways of IPF in the Gene Expression Omnibus database were analyzed, and CAT was molecularly docked with the corresponding key proteins to screen its pharmacological targets, which were then verified using an animal model. The results show that collagen metabolism imbalance, inflammatory response, and epithelial-mesenchymal transition (EMT) are the core processes in IPF, and the TGF-β1/Smad3 and Wnt/β-catenin pathways are the key signaling pathways for the development of pulmonary fibrosis. Our results also suggest that CAT binds to TGF-βR1, Smad3, Wnt3a, and GSK-3β through hydrogen bonds, van der Waals bonds, and other interactions to downregulate the expression and phosphorylation of Smad3, Wnt3a, GSK-3β, and β-catenin, inhibit the expression of cytokines, and reduce the degree of oxidative stress in lung tissue. Furthermore, CAT can inhibit the EMT process and collagen remodeling by downregulating fibrotic biomarkers and promoting the expression of epithelial cadherin. This study elucidates several key processes and signaling pathways involved in the development of IPF, and suggests the potential value of CAT in the treatment of IPF.

Download Full-text

5-Aminosalicylic acid Attenuates Paraquat-induced Lung Fibroblasts Activation and Pulmonary Fibrosis of Rats

10.21203/rs.3.rs-238806/v1 ◽

2021 ◽

Author(s):

Hui Chen ◽

Jinfeng Cui ◽

Juan Wang ◽

Yuan Wang ◽

Fei Tong ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Signaling Pathway ◽

Human Lung ◽

Lung Fibroblasts ◽

Control Group ◽

Peroxisome Proliferator ◽

Aminosalicylic Acid ◽

Human Lung Fibroblasts ◽

Group 5 ◽

Tgf Β1

Abstract Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which becomes the focus of treatment. More and more studies have found that 5-Aminosalicylic acid (5-ASA) may be a prospective therapy against fibrotic diseases. In the present study, we observed whether 5-ASA could attenuate the pulmonary fibrosis in PQ-treated rats and human lung fibroblasts (WI38VA13) cells, and subsequently explored the possible underlying mechanisms. Wistar rats were divided into control group, 5-ASA group, PQ group and PQ + 5-ASA group. Rats were sacrificed on 3, 7, 14, and 28 days after PQ treatment. We observed pulmonary histopathological changes and fibrosis formation among different groups through hematoxylin and eosin (H&E) and Masson staining and TGF-β1, p-Smad3 and the peroxisome proliferator activated receptor γ (PPARγ) pulmonary content via immunohistochemical staining and Western blot. In addition, human lung fibroblasts WI38VA13 were also divided into control group, PQ group, 5-ASA group and PQ + 5-ASA group. And the role of TGF-β1 signaling pathway regulated factors (TGF-β1, p-Smad3 and PPARγ) were explored. Treatment with 5-ASA significantly inhibited the PQ-induced activation of TGF-β1 signaling pathway in human lung fibroblasts WI38VA13 cells. In conclusion, the results of this study suggested that 5-ASA has potential value in the treatment of PQ-induced pulmonary fibrosis via suppressing the activation of TGF-β1 signaling pathway.

Download Full-text