The effect of compound essential oil on PM2.5 induced endothelial mesenchymal transition of mouse pulmonary microvascular endothelial cells
PM2.5 (fine particulate matter with aerodynamic diameter ≤ 2.5 μ m) is one of the main pollutants causing air pollution. PM2.5 has a large specific surface area, making it easy to carry toxic and harmful substances that enter the deep alveoli through the respiratory system, causing lung inflammation and damage and potentially progressing to pulmonary fibrosis. The significant increase in fibroblasts is the main characteristic of pulmonary fibrosis. Collagen accumulates in the periphery of pulmonary blood vessels and alveolar septa, and fibrous connective tissue also increases accordingly, ultimately leading to an increase in matrix hardness and difficulty in gas exchange. Endoendothelial mesenchymal transition (EndMT) is one of the sources of fibroblasts, characterized by the gradual loss of phenotype markers such as vascular endothelial cell cadherin (VE cadherin/CDH5) and platelet endothelial cell adhesion molecule-1 (CD31/PECAM-1) in endothelial cells, and increased expression of phenotype markers such as type I collagen (Col1), vimentin, alpha smooth muscle actin (cta2/alpha SMA) in mesenchymal cells. Fibroblast specific protein 1 (Fsp1/S100A4) and other proteins. During this process, the proliferation and migration abilities of cells gradually increase, ultimately leading to changes in cell morphology. In recent years, the molecular mechanism of inflammation induced EndMT has been understood. The TGF – β pathway is a classic pathway for inflammation induced EndMT, and all three isoforms of TGF – β can mediate the occurrence of EndMT in various cell lines, with TGF – β 1 playing the most significant role. Previous studies have shown that PM2.5 can enhance lung inflammation by activating the TGF – β signaling pathway, but the specific mechanism of action and its impact on pulmonary fibrosis are still unclear.

Essential oils (EOs) can be extracted from the flowers, fruits, seeds, leaves, stems, or roots of aromatic plants through distillation, extrusion, or solvent extraction. They have various activities such as antibacterial, anti-inflammatory, antioxidant, anti-aging, and have been widely used in clinical, pharmaceutical, health and wellness fields. The “natural therapy” that uses essential oils extracted from aromatic plants for therapeutic medical purposes is called aromatherapy, which can be administered through external methods such as aromatherapy, inhalation, topical application, or hydrotherapy. Eucalyptus, peppermint, frankincense, and spruce essential oils are common essential oils used to treat respiratory diseases. They can dissolve mucus, remove phlegm, kill bacteria, relieve cough, and spasms, thereby protecting the lungs from inflammation and relieving lung inflammation. We used aromatherapy to compound the four types of essential oils mentioned above into a compound essential oil for this experiment, and explored the intervention effect of the compound essential oil on PM2.5 induced EndMT through the TGF – β 1/SMAD3/p-SMAD3 signaling pathway.

PM2.5 is one of the most concerning air pollutants. Due to its small particles, it is easier to reach the alveoli and blood of the human body, thereby accelerating the disease progression of those who have already suffered from lung diseases. The endothelial cells on the inner wall of blood vessels come into direct contact with blood and exhibit significant heterogeneity in response to foreign body stimulation in the blood. Endothelial cells in the EndMT process are one of the sources of fibroblasts, which not only leads to a significant increase in fibroblasts, but also reduces capillary bed density, exacerbates tissue hypoxia and ischemia, and accelerates the process of pulmonary fibrosis. Preliminary laboratory experiments have shown that PM2.5 can induce EndMT in MHC cells. The protein expression levels of Acta2, Col1, and S100A4 increase, while the expression levels of CD31 and CDH5 decrease, indicating that PM2.5 can induce EndMT in mouse pulmonary microvascular endothelial cells. The increased protein expression level of S100A4 also confirms the important role of End MT in pulmonary fibrosis.

The main components of the compound essential oil used in this experiment are alpha pinene (16.54%), eucalyptol (16.7%), and menthol (12.86%). Eucalyptol is a tumor necrosis factor alpha repressor that can effectively inhibit the progression of asthma and chronic pulmonary obstructive diseases, and has potential as a steroid anti-inflammatory drug. Alpha pinene has antioxidant and antibacterial effects, and exhibits certain inhibitory activity against tumor cell line A549. Menthol can block the L-type calcium channels in vascular smooth muscle, which has a certain therapeutic effect on cardiovascular diseases. This experiment found that PM2.5 can induce the proliferation and migration of MHC cells to reach their peak after 48 hours of treatment. However, in the compound essential oil group, the overall function of MHC cells did not undergo significant changes due to PM2.5 induction. After 36 hours of intervention with compound essential oil, the protein expression levels of Acta2 and Col1 were significantly reduced, indicating that compound essential oil has a good alleviating effect on the occurrence of EndMT.

Research has found that TGF – β plays an important role in the development, injury, and repair of the lungs. It can promote the synthesis of extracellular matrix by activating TGF – β 1 receptors and regulate the pathogenesis of pulmonary fibrosis. Previous experiments have found that after treating MHC cells with PM2.5 for 48 hours, the protein expression levels of TGF – β 1, TGF – β R1, and p-SMAD3 increase. However, after adding SB431542 to the PM2.5 group for 48 hours, the expression of TGF – β R1 and p-SMAD3 decreases. As SMAD protein phosphorylation is a typical indicator of the TGF – β 1 signaling pathway, it can be seen that PM2.5 can activate TGF – β R1 by regulating the TGF – β signaling pathway and stimulating the transformation of TGF – β 1 into an active form. TGF – β R1 is activated and binds to SMAD3. Overexpression of p-SMAD3 can promote the expression of mesenchymal cell markers and collagen accumulation. In the compound essential oil group, the protein expression levels of TGF – β R1 and p-SMAD3 were significantly reduced. The compound essential oil inhibited the activation of TGF – β R1 to decrease the expression of p-SMAD3, thereby suppressing PM2.5 induced EndMT.

It can be inferred that compound essential oils can slow down the occurrence of EndMT induced by PM2.5 in pulmonary endothelial cells by inhibiting the expression of the TGF – β 1/SMAD3/p-SMAD3 signaling pathway. These findings provide a theoretical basis for the repair of EndMT damage in the lungs after exposure to PM2.5 and the intervention plan of compound essential oils.