The two main findings of this study are as follows: (1) Quercetin can significantly improve the hemodynamic changes, right ventricular remodeling and pulmonary vascular remodeling in MCT induced PAH rats; (2) Quercetin inhibits the proliferation, migration and phenotypic transformation of pulmonary artery smooth muscle cells by regulating TGF-β1/Smad2/Smad3 pathway, thus playing a protective role in PAH pulmonary vascular remodeling.
PAH is a rare progressive disease, which refers to a cardio pulmonary vascular disease characterized by pulmonary vascular remodeling due to the progressive increase of the average pulmonary artery pressure, and ultimately leads to right heart failure and death[4, 55]. As early as 1961, some scholars found that MCT extracted from the seeds of the pig excrement bean plant can increase the pulmonary artery pressure by inducing pulmonary vascular disease, which is widely used in the establishment of animal models of PAH disease[56]. Therefore, the PAH animal model was established by intraperitoneal injection of MCT in this experiment. After 28 days of intraperitoneal injection of MCT, the results of Doppler ultrasound showed that: the notch and peak value of pulmonary vessels in rats moved forward in the middle of contraction, PAAT decreased and mPAP increased; HE staining showed that the right ventricular wall thickness increased and RVHI% increased, indicating that the right heart function of rats in MCT group was not complete; The stenosis of the lumen of pulmonary small and medium-sized arteries is accompanied by thickening of the wall, disordered cell arrangement, and deposition of exogenous matrix, which indicates that pulmonary vascular remodeling is obvious. The above results indicate that the PAH model has been successfully established.
Quercetin, as one of the flavonol compounds existing in many plants, has a variety of biological activities, including anti-inflammatory, antioxidant, antifibrosis, inhibition of cell proliferation, migration and so on[57–59]. Some studies have shown that quercetin has a protective effect on PAH rats, but its specific mechanism is still unclear[22, 60]. Research shows that pulmonary vascular remodeling plays an important role in the occurrence and development of PAH[61, 62]. At the animal level, SD rats were injected MCT intraperitoneally and administered quercetin for 14 days. The ultrasound results showed that PAAT increased and mPAP decreased, indicating that quercetin could effectively reduce the pressure of pulmonary artery wall; At the same time, the thickness of the right ventricle and the function of the right heart in rats after intragastric administration of quercetin decreased, and the wall thickness and the area of the lumen of the pulmonary arterioles decreased, and the lumen increased. The above results indicate that quercetin can effectively inhibit the mean pulmonary artery pressure, right ventricular remodeling and pulmonary arteriole remodeling in MCT induced PAH rats.
Pulmonary vascular remodeling is an important pathophysiological change of PAH, in which the proliferation, migration and phenotypic transformation of PASMCs in the vascular media is one of the key events of pulmonary vascular remodeling. It has been pointed out that PDGF-BB, as one of the super mitogens, has a variety of cell population proliferation capabilities, and can promote the proliferation and migration of PASMCs in cell experiments. In addition, there are more mitochondria and rough endoplasmic reticulum in PASMCs after the action of PDGF-BB, and the cells are converted from contraction to synthesis phenotype, and the collagen content is more abundant[63, 64]. Therefore, PDGF-BB was selected as the cell modeling drug at the cell level. The proliferation and migration of HPASMCs were tested by EDU, scratch and Transwell TM experiments. The results showed that quercetin could inhibit the proliferation, scratch healing and migration of HPASMCs, suggesting that quercetin might have a protective effect on PAH induced pulmonary vascular remodeling by inhibiting the proliferation and migration of HPASMCs.
According to research, pulmonary vessels have strong plasticity. When the vessels are stimulated by pathophysiology such as endometrial damage, excessive blood flow, inflammatory factors, ischemia, hypoxia, dust, and NO, the cell phenotype changes from a highly differentiated contraction phenotype to a poorly differentiated synthetic phenotype, such as the expression of α-SMA and SM22a proteins decreases, and the expression of OPN and epirelin proteins increases[65]. Some studies have shown that PDGF-BB, as an important factor to stimulate cell proliferation, can induce PASMCs to undergo phenotype transformation by promoting the expression of α-SMA protein in cells[66]. We used immunofluorescence, immunohistochemical staining and Western Blot to detect the expression of phenotypic transformation related proteins through animal experiments. The experimental results showed that quercetin could significantly reduce the expression of OPN protein in pulmonary vessels and inhibit the overexpression of α-SMA protein. Cell experiments further proved that quercetin had an inhibitory effect on the phenotypic transformation of HPASMCs. The review showed that quercetin could inhibit the transformation of cells from contractile to synthetic types, to some extent, the remodeling of pulmonary vessels was reversed.
As a key protein of abnormal cell proliferation, PCNA has a certain correlation with the occurrence and development of cell proliferation diseases. As a migration related protein[33, 67], OPN affects cell migration and the metabolism of cytoskeletal proteins in extracellular matrix. Changes in a variety of cancer cells and smooth muscle cells suggest changes in cell migration capacity[68, 69]. Therefore, in this study, we detected the expression of proliferation and migration related proteins in each group at the animal level. The results showed that quercetin had a certain inhibitory effect on the proliferation and migration related proteins PCNA and OPN in the pulmonary vessels of PAH rats. The corresponding protein changes were also detected at the cell level, and the results were consistent with animal experiments. Quercetin also had a significant inhibitory effect on the mRNA expression of proliferation and migration related proteins at the cell level. To sum up, quercetin can effectively inhibit the expression of proliferation and migration related proteins of HPASMCs, and reverse the remodeling of pulmonary vessels to a certain extent, which is consistent with the results of HE in small and medium-sized pulmonary arteries and the EDU, scratch and Transwell™ experiments in cells in the above animal experiments.
TGF-β1-Smad2/Smad3 signaling pathway, as a member of TGF-β superfamily, plays different roles in cell proliferation, migration, differentiation, adhesion, death and other processes, as well as in embryonic development, formation of extracellular matrix, bone formation and reconstruction and other physiological processes[70, 71]. Some studies have shown that in the classic TGF-β1 pathway, TGF-β1 can combine with TbRⅠ and TbRⅡ receptors on the cell surface to form trimer complexes, which can phosphorylate Smad2/3 itself in the cell and form complexes with Smad4 to promote nuclear translocation, and enter the nucleus to start the synthesis and secretion of factors related to proliferation and migration[72]. Therefore, TGF-β1-Smad2/Smad3 signaling pathway plays an indispensable role in the process of pulmonary artery remodeling[15, 73]. In order to explore how quercetin inhibits pulmonary vascular remodeling, is it through TGF-β1-Smad2/Smad3 signaling pathway? We examined the effects of quercetin on TGF-β1-Smad2/Smad3 pathway at animal and cell levels, respectively. The results showed that quercetin could effectively inhibit the levels of TGF TGF-β1 and P-Smad2/Smad3 proteins in pulmonary vessels of MCT induced PAH rats, but did not affect the expression of the total Smad2/Smad3 protein; This result was verified at the cell level, and quercetin did not significantly affect the mRNA levels of Smad2 and Smad3, indicating that quercetin plays a role in nuclear regulation by inhibiting the expression of TGF-β1 and then inhibiting the phosphorylation of Smad2 and Smad3.
Our previous research showed that HPASMCs can promote the phosphorylation of Smad2 and Smad3 proteins in cells, promote the expression of PCNA and OPN related to cell proliferation and migration, and inhibit the expression of α-SMA, a cell contraction phenotype protein, under the effect of TGF-β1. After the TGF-β1 inhibitor SB431542 was used, the expression of TGF-β1 was inhibited, the expression of downstream P-Smad2 and P-Smad3 was reduced, and the expression of proteins related to proliferation, migration and phenotype transformation in HPASMCs was inhibited, thus inhibiting cell proliferation, migration and promoting cell transformation from synthetic type to contractile type, the healing ability and migration ability of cells were also inhibited[16]. Combined with our experimental results, it can be concluded that quercetin may affect the proliferation, migration and phenotype transformation of HPASMCs by regulating TGF-β1-Smad2/Smad3 signaling pathway.
To sum up, through the establishment of the SD rat PAH model in vitro and the cultivation of HPASMCs model in vitro, this topic proved that quercetin can inhibit the proliferation and migration of pulmonary artery smooth muscle cells by regulating TGF-β1-Smad2/Smad3 pathway, thereby inhibiting pulmonary vascular remodeling, and playing a protective role on PAH.