Integrating fecal metabolomics and intestinal microbiota to study the mechanism of cannabidiol in the treatment of idiopathic pulmonary fibrosis

Abstract

1 Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive pulmonary interstitial disease with unknown causes, mainly occurring in middle-aged and elderly people. The prognosis is poor, and the median survival is only 3–5 years. The main feature of IPF is tissue scarring caused by excessive deposition and excessive repair of extracellular matrix (ECM) (Mei et al., 2022). It is currently recognized that pulmonary fibrosis is an epithelial-driven disease, the main mechanism of which is epithelial-mesenchymal transition (EMT). Sustained epithelial cell injury would cause abnormal activation of epithelial cells and then secrete a large amount of transforming growth factor-β (TGF-β) (Inui et al., 2021), which was characterized by interstitial scar formation and irreversible decline in lung function (Strykowski and Adegunsoye, 2023). Smoking, air pollution, and occupational exposure are considered to be risk factors for IPF (Schäfer et al., 2020). Data show that pulmonary fibrosis affects about 3 million people in the world, and with aging and serious air pollution, global morbidity and mortality are increasing year by year (Moss et al., 2022). At present, Nidanib and pirfenidone have been approved by the FDA and are clinically used to treat pulmonary fibrosis, but they can only delay the fibrosis process, and the prognosis is still poor, and long-term use will cause gastrointestinal and other adverse reactions (Spagnolo et al., 2018). Therefore, it is necessary to further study the physiological and pathological mechanisms of pulmonary fibrosis and actively seek new treatment strategies.

Cannabidiol (CBD) is a non-psychoactive derivative of the cannabis plant (Meissner and Cascella, 2023) and has been valued for its anti-anxiety, anti-emetic, anti-inflammatory, and anticancer properties (Legare et al., 2022). As a powerful antioxidant, CBD acts on a variety of receptor sites, directly or indirectly causing a wide range of anti-inflammatory and immunomodulatory effects (Atalay et al., 2019). In 2018, the FDA approved the use of CBD to treat epilepsy in children, especially Dravet syndrome (Vossler et al., 2018; Reddy, 2023). Previous research has shown that CBD can reduce the production of related cytokines in animal models of chronic asthma. Recently, it has been established that CBD is involved in regulating inflammatory responses, including inflammatory lung diseases, and has a positive effect on acute and chronic inflammation (Eskander et al., 2020; Urits et al., 2020; Porter et al., 2021). Previous studies of our research group found that CBD can reduce the expression of TGF-β and α-SMA in rat lung tissue; the content of TGF-β1, α-SMA, and NF-κB p65m RNA in lung tissue was decreased, and the expression level of Nrf2 m RNA was increased, especially in the high-dose group (Sun et al., 2023). In addition, CBD is well tolerated with no significant side effects, and it may also be an effective candidate for the treatment of pulmonary fibrosis. However, there is little research on the pharmacological activity and mechanism of CBD in the treatment of IPF.

There is evidence that IPF is inextricably linked to the gut microbiome, which is considered an indispensable “metabolic organ” that plays a crucial role in maintaining human health or causing disease (Chen et al., 2021). Significant changes in gut microbiota composition have been observed in IPF patients, and restoring gut microbiota imbalance through dietary probiotic inulin supplementation with alpha or linolenic acid-rich flaxseed oil can help improve IPF (Abidi et al., 2020). Previous studies have suggested that immune cells and cytokines, which are prompted by the gut microbiota and its metabolites, including SCFAs, may penetrate the systemic circulation via the blood and lymphatic system. This process plays a crucial role in controlling immune and inflammatory reactions within the lungs, thereby impacting respiratory wellbeing and ailments (Ashique et al., 2022). On the other hand, the imbalance of intestinal microbiota can stimulate inflammatory activity and disrupt energy balance, affecting respiratory immunity and barrier function (Chunxi et al., 2020). Likewise, specific plant life can aid in the body’s maintenance of a balanced immune system by diminishing the inflammatory response and assisting the lungs in combatting infection (Ma et al., 2022).

Sequencing of the 16S rRNA gene enables precise identification of the structure of gut microorganisms. Metabolomics offers a comprehensive assessment of changes in metabolites during the occurrence and progression of diseases. The utilization of 16S rRNA gene sequencing and metabolomics has become widespread in exploring the mechanisms of diseases and drug therapies, unraveling the pathogenic processes, and identifying disease biomarkers.

Consequently, in conjunction with fecal metabolomics and gut microbiota analysis, this current investigation more comprehensively illustrates CBD’s protective mechanisms in IPF rat models (Figure 1). The findings of this study provide experimental evidence and scientific support for the lung-protective effects of CBD, as well as offer fresh perspectives for the rational development and utilization of CBD resources in the pharmaceutical and food sectors.

2 Materials and methods

3 Results

4 Discussion

In this study, BLM was used to establish IPF model rats. BLM is a chemotherapy medication that disrupts the normal cell division process, leading to the generation of excessive amounts of unstable molecules known as free radicals. This, in turn, triggers inflammatory responses and causes damage to the lung tissues, including fibroblast activation and subsequent fibrosis. Consequently, BLM is extensively employed in the creation of animal models that mimic IPF (Della Latta et al., 2015; Song et al., 2023). The deposition of extracellular matrix (ECM) is a fundamental pathological characteristic of IPF. Among the various components that make up the ECM, collagen is particularly prominent (Habermann et al., 2020). Furthermore, hydroxyproline, a constituent of collagen, plays a crucial role in its composition (Cao et al., 2022). Through the utilization of H&E and Masson staining techniques, we observed pronounced fibrosis, structural impairment, and the presence of elongated fiber bands in the lungs of rats in the model group. Additionally, biochemical analysis demonstrated a notable elevation in the levels of hydroxyproline. Prednisone, a hormone-based pharmaceutical commonly used for the clinical treatment of IPF (Kooistra et al., 2023), was employed as the reference drug in numerous previous animal studies investigating IPF. Our findings indicate that there was no significant disparity between the high-dose CBD group and the positive control group in terms of their capacity to enhance lung permeability and ameliorate the pathological alterations observed in rats with IPF. This suggests that CBD possesses the therapeutic potential for the treatment of IPF.

Furthermore, IPF (Moss et al., 2022; Savin et al., 2022) is characterized by persistent inflammation, which is a crucial pathological alteration. Fibroblasts undergo constant activation and release substantial quantities of extracellular matrix (ECM) during chronic inflammatory conditions, ultimately resulting in fibrosis and the degradation of regular alveolar structures. Past investigations have indicated the significant involvement of macrophages in IPF (Kishore and Petrek, 2021). In the presence of inflammation, macrophages can swiftly migrate toward the site of inflammation, release numerous inflammatory mediators, and stimulate fibroblast activation and replication (Buechler et al., 2021; Du et al., 2022). ELISA results showed that compared with the control group, there were a large number of pro-inflammatory cytokines in the serum of IPF model rats, which was consistent with the results of other studies (Qin et al., 2021; Chen et al., 2022). The results of the study showed that CBD reduced the inflammatory response in IPF rats.

Oxidative stress is one of the important pathogenesis of IPF (Otoupalova et al., 2020; Forman and Zhang, 2021). This study assessed lung oxidative stress by measuring oxidative stress-related enzyme activities and levels of peroxidation markers. The findings revealed that the model group had a decrease in SOD activity, an enzyme responsible for combating oxidative stress, and an increase in MDA level, a marker of peroxide, suggesting that the lungs experienced severe oxidative stress. However, our study discovered that CBD could improve the antioxidant ability of rats with IPF, thereby reducing damage to lung tissues caused by the peroxidation of lipids.

Metabolomics analysis showed that CBD-H regulates 17 key biomarkers in the IPF model, including Linoleate; Sphingosine; Sphinganine; Sphingomyelin; Phosphatidylethanolamine; LysoPC(20:4/0:0); α-Linolenic acid, etc. These metabolites are related to linoleic acid metabolism, glycerol phospholipid metabolism, linolenic acid metabolism, sphingolipid metabolism, and other pathways, and may inhibit the progression of pulmonary fibrosis. Linoleic acid participates in the formation of phospholipids and maintains the fluidity of the cell membrane. The linoleic acid released by phospholipids in cell membranes can form metabolites involved in cell signaling pathways through the action of various enzymes. Linoleic acid is a precursor to arachidonic acid, which produces prostaglandins and leukotrienes with inflammatory potential (Sonnweber et al., 2018; Wang et al., 2019). The results suggest that CBD may improve pulmonary fibrosis and maintain homeostasis by slowing down linoleic acid metabolism.

Lipids are composed of a variety of molecules that play a key role in cellular energy storage, structure, and signaling (Brohée et al., 2021). Plasma plays an indispensable role in immune response by influencing immune response through the release of lipid-derived mediators (Balboa et al., 2019). In recent years, more and more attention has been paid to the role of lipids in lung and respiratory diseases, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease, all of which are associated with metabolic abnormalities, such as research results demonstrating changes in glycerophospholipid metabolism in IPF patients (Yan et al., 2017). Glycerophospholipids are the main components of biofilms and play a crucial role in the normal function of cells (Powers and Trent, 2019). Glycerol phospholipid metabolism produces a variety of metabolites, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (Lyso PE), lysophosphatidylcholine (Lyso PC), etc. The changes of these metabolites will also affect the normal metabolism (Stephenson et al., 2019). Lyso PC is a precursor of lysophosphatidic acid, which can induce pulmonary fibrosis by mediating vascular leakage and fibroblast migration and proliferation (Corte et al., 2021). Ethanolamine undergoes phosphorylation by ethanolamine kinase, resulting in the formation of phosphoethanolamine (Lykidis et al., 2001). Subsequently, phosphoethanolamine and phosphocholine cytidylyl transferase collaborate to generate high-energy donor CDP-ethanolamine (Fullerton et al., 2007), while 1, 2-diacylglycerol ethanolamine phosphotransferase utilizes the energy supplied by CDP-ethanolamine to bind ethanolamine to the diacylglycerol in the membrane, leading to the formation of PE (Calzada et al., 2016). PE underwent three successive methylation reactions to generate PC, which is called the PE methyltransferase pathway (Ridgway and Vance, 1987). PC can also generate CDP-choline through choline phosphorylation and choline cytidylyl phosphate transferase, and 1, 2-diacylglycerol choline phosphotransferase catalyzed the exchange of cytidine 5′-monophosphate for diacylglycerol formation, a process known as the Kennedy pathway (Sonkar et al., 2019). PC, as the most abundant phospholipid in animals, is involved in several important metabolic pathways, and abnormal levels of PC have been found in many cancer diseases. For example, the hydrolysis of PC produces lipid mediators. The production of these lipid mediators is beneficial to the survival, proliferation, and growth of cancer cells. Immunomodulatory cell-to-cell crosstalk ultimately leads to resistance to therapy (Saito et al., 2022). The alveoli, the most active lipid metabolizer in the lung, requires surfactant lipids for the synthesis of respiration (Agudelo et al., 2020), and PC and PE are the key lipids of pulmonary surfactants, and the total saturation of silicosis rat lungs increases (Srivastava and Misra, 1986). Xia et al. (2021) also confirmed that the contents of PC and PE in bleomycin-induced pulmonary fibrosis rats were increased. This is consistent with the results of this study. After CBD treatment, LysoPC(18:0/0:0), LysoPC(16:0/0:0), PE, Lyso PC(20∶4/0∶0), LysoPC[18:1(9Z)/0:0] were significantly decreased compared with the model group. Therefore, it is speculated that CBD may reduce the synthesis of PC by slowing down the PE methyltransferase pathway and the Kennedy pathway in glycerophospholipid metabolism, and regulate inflammation and endothelial cells. Proliferation and apoptosis, thus affecting the development of IPF. α-linolenic acid has been reported to have protective effects on cardiovascular nerves, anticancer, anti-osteoporosis, anti-inflammatory and antioxidant effects (Yuan et al., 2022). Studies have found that flaxseed oil can effectively protect rat lung tissue from bleomycin-induced pulmonary toxicity, promote increased lumen patency and decreased pulmonary septal thickness, reduced inflammatory cell infiltration, delayed edema formation, reduced vascular inflammation and pulmonary and peribronchial fibrosis (Lawrenz et al., 2012).

Extensive research has been conducted on the potential involvement of the gut microbiota in IPF, which has demonstrated a significant correlation with inflammation (Bhattacharya et al., 2022). This study employed 16S rRNA sequencing analysis to ascertain the impact of CBD on the structure and composition of intestinal microbiota in IPF rats.

This article further elucidates the changes in the intestinal microbiome of the IPF model after CBD-H intervention to identify the microbiome associated with IPF progression, and the top few bacteria with the highest relative total abundance at the genus level were selected for comparison. The IPF rats exhibited a significant decrease in the relative abundance of [Eubacterium]_coprostanoligenes_group, Prevotella, Clostridia_UCG-014, Ruminococcus, Fusobacterium, Streptococcus and Collinsella while witnessing a notable increase in the relative abundance of Lachnospiraceae_NK4A136_group and Pseudomonas. CBD may significantly increase Prevotella, Clostridia_UCG-014, Ruminococcus, Fusobacterium, Streptococcus, and Collinsella and decrease the abundance of Lachnospiraceae_NK4A136_group and Pseudomonas. Prevotella is often considered a bacterium associated with a healthy plant-based diet, acting as a “probiotic” in the human body. The decline of Prevotella has been associated with certain diseases. Ruminococcus produces metabolites in the form of glucomannan polysaccharide. Ruminalcoccal polysaccharides can activate immune system cells, including tumor necrosis factor (TNF-α), while ruminal bacillus plays a crucial role in the synthesis of single-chain fatty acids (SCFAs), which serve as the primary energy source for colon cells. Patients with end-stage renal disease experience a substantial decrease in the production of SCFAs by bacteria (Zhu et al., 2020). Yu et al. (2021) discovered a positive association between Rumenococcaceae and liver health status, while a negative association was observed with factors related to injuries. Furthermore, Ruminococcaceae may impede the progression of chronic kidney disease (Hsiao et al., 2021). Consistent with the results of this article, Ruminococcus can be used for further research as a pathogenic bacterium. However, there is debate about the effects of Lachnospiraceae on the respiratory system. Some studies have reported the protective effect of Lachnospiraceae in the respiratory system to a certain extent. Zhao et al. (2021) found in the study of particulate matter-induced lung injury in mice that the Lachnospiraceae_NK4A136_group may be the core intestinal microbe playing a protective role. However, other studies have noted that there is no negative association between Lachnospiraceae and respiratory diseases. Li et al. (2019) found that an increased abundance of Lachnospiraceae_NK4A136_group was associated with high levels of IL-17, which is known to promote inflammation by activating CD4 ⁺ T cells. Furthermore, there was a significant positive correlation between the Lachnospiraceae_NK4A136_group and IgE and IL-33 (Wang et al., 2022). The results of this experiment showed that the relative abundance of Lachnospiraceae_NK4A136_group in the intestinal microbiota of IPF mice was significantly increased and reversed by CBD intervention. This appears to be at odds with prior findings, and the discrepancies between the studies may be due to disparities in environment, geography, and dietary practices. Furthermore, varying sampling techniques may also be a contributing factor to the disparate outcomes. Furthermore, it is linked to the diversity of each individual’s pulmonary microbiome. Hence, it is imperative to delve deeper into the precise function of the Lachnospiraceae_NK4A136_group in the context of lung disease in subsequent research. In addition, CBD increases the abundance of beneficial bacteria, such as Fusobacterium, Streptococcus, and Collinsella. In summary, CBD can effectively inhibit inflammatory cell infiltration and pulmonary fibrosis by regulating intestinal microbiota.

What is the mechanism by which CBD affects intestinal microbes and alters the metabolic composition of the host stool? It is widely acknowledged that small molecule metabolites play a crucial role in linking the gut-lung axis. This article conducted a correlation analysis between intestinal microbiota and potential metabolic markers and found that there is a significant correlation between the two. CBD can control the levels of Lachnospiraceae_NK4A136_group, Pseudomonas, Clostridia_UCG-014, Collinsella, Prevotella, [Eubacterium]_coprostanoligenes_group, Fusobacterium, Ruminococcus, and Streptococcus, thereby reinstating the activity of gut bacteria and reversing the pattern of fecal metabolism. Simultaneously, it has the ability to activate Sphinganine, Sphingomyelin, alpha-Linolenic acid, Sphinganine, Linoleic acid, Diminish Phosphatidylethanolamine and PC (20:4/0:0), control the metabolism of linoleic acid, glycerol phospholipid, linolenic acid and sphingolipid, shield lung tissue from the consequences of inflammatory elements and ultimately act as an anti-IPF agent.

5 Conclusion

In conclusion, this study suggests that CBD treatment can improve pulmonary fibrosis by inhibiting inflammatory responses and oxidative stress. This research employed a combination of pharmacodynamics, 16SrRNA, and metabolomics to investigate the impact of CBD on pulmonary fibrosis, which provided a basis for further exploration into the pathology of IPF and the creation of new pharmaceuticals. Future research could employ targeted metabolomics to ascertain the metabolic control mechanisms of CBD on pulmonary fibrosis. Furthermore, due to the strong correlation between metabolic alterations and the development of pulmonary fibrosis, such as inflammation and oxidative stress, further research can be done with in vitro models to gain a better understanding of how CBD hinders inflammation and oxidative stress by influencing metabolism.

Acknowledgments

Funding Statement

The authors declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Talent Training Project of Central Support for Reform and Development of Local Universities in Heilongjiang Province (No. 2021ZYQGLG001).

References