P38 kinase is a proline-mediated serine/threonine kinase of the mitogen-activated protein kinase (MAPK) family, which is activated by environmental stress and signal pathways. P38MAPKs (especially P38-a) are involved in cellular responses to stress, environmental and intracellular stresses at many levels, such as high osmotic pressure, oxidative stress, inflammation, DNA damage and other physiological conditions that involve cell changes (7). P38 -a is involved in a variety of functions, and the disorder of this pathway is related to diseases such as inflammation, immune disorders or cancer (8). The activation of P38 is mediated by the phosphorylation of specific regulatory tyrosine and threonine sites, and the three kinases MKK3, MKK4, and MKK6 are the upstream activators of P38(9). P38a regulates many functions of cardiomyocytes, including hypertrophy, contractility, fibrosis and apoptosis (10,11༌12).
Atrial muscle fibrosis leads to local conduction slowdown and conduction disorders, leading to unidirectional conduction block (13). Fibrosis increases the number of fibroblasts and change their characteristics, by changing the interaction between cardiomyocytes and fibroblasts to couple the electrophysiological behavior of cardiomyocytes, thereby promoting atrial fibrillation (14). The role of the RAAS system in the remodeling of atrial structure has been confirmed. Clinical studies have shown that RAAS inhibitors are benefit for patients with atrial fibrillation after pulmonary vein isolation (15). RAAS inhibitors also inhibit atrial fibrosis and atrial remodeling, and delay atrial fibrillation (16, 17). Ang II is the central factor of RAAS, which causes vasoconstriction, increases myocardial afterload, promotes left ventricular hypertrophy, indirectly increases atrial pressure, and increases the ductility of myocardial cells (18). In addition, Ang II increases oxidative stress, thereby inducing inflammatory collagen fiber deposition and causing atrial fibrosis. In animals with an overexpression of angiotensin converting enzyme, Ang II levels are significantly increased, and there was obvious atrial enlargement, atrial fibrosis and atrial fibrillation. Ang II promotes atrial remodeling through TGF-β/Smad2/3 signaling pathway (19). MAPKs are a group of important downstream molecules of Ang II, which are involved in the increased expression of TGF-β1 inducing by Ang II (20). The increased expression of TGF-β1 inducing by Ang II is the main mechanism of Ang II inducing atrial fibrosis. In a clinical study, 56 patients with rheumatic heart disease are divided into atrial fibrillation group and sinus rhythm group. Left atrial appendage tissue is collected during cardiac surgery to assess myocardial fibrosis. The study found that the atrial MAPKs activity in the atrial fibrillation group is significantly higher than sinus rhythm group, the atrial TGF-β1 and CTGF mRNA and protein expression in patients with atrial fibrillation increases significantly, confirming that the MAPKs/TGF-β1/TRAF6 signaling pathway is involved in the occurrence of atrial fibrosis in patients with atrial fibrillation (21༌22). In addition, the increased expression of TGF-β1 induced by Ang II is the main mechanism of Ang II-induced myocardial fibrosis (23). Ang II increased the activation of mouse fibroblast MAPKs, up-regulate the expression of TGF-β1 and CTGF, and promote the proliferation of fibroblasts (24). The increase in CTGF expression induced by Ang II or TGF-β1 is an important factor involved in atrial fibrosis and atrial fibrillation. In vitro studies on mouse fibroblasts show that Ang II stimulates TGF-β1 on atrial fibroblasts, which acts in a Smad-independent manner. In this study, it is also confirmed that the TGF-β1/TRAF6 signaling pathway is involved in atrial fibrosis.
Transforming growth factor (TGF-β1) is a key fibroblast growth factor. TGF-β1 regulates cell proliferation, apoptosis and migration, and regulate the synthesis of extracellular matrix (such as up-regulating the expression of fibronectin and collagen fibers). Overexpression of TGF-β1 may cause atrial fibrosis and atrial fibrillation. The increase in CTGF expression induced by Ang II or TGF-β is an important factor involved in atrial fibrosis and atrial fibrillation (25). TGF-β may be the main switch that regulates the transition from inflammatory response to fibrosis (26). Masaki Ikeuchi et al. show that early inhibition of TGF-β aggravates ventricular dysfunction and inflammatory response, while late destruction of TGF-β signaling protects interstitial fibrosis and hypertrophic remodeling (27). Peter P R et al. show that although extensive inhibition of TGF-β after infarction lead to early death of heart rupture, the specific destruction of TGF-β receptors by cardiomyocytes has a protective effect and extensively stimulates anti-inflammatory and cytoprotective signals (28). Therefore, the adverse effects of early TGF-β inhibition on infarcted myocardium may not be due to the direct effect of cardiomyocyte survival, but reflect the loss of anti-inflammatory effects of inflammatory cells, endothelial cells or fibroblasts(29).
This study confirmed that inflammatory stress triggers the MKK-P38MAPKs signaling pathway, which increased the expression of MKK3, MKK4, MKK6, and PMKK4 proteins, and the expression of phosphorylated PP38 protein was also significantly increased, and the concentration of inflammatory factors TGFβ also increased. Thus, these changes may cause myocardial fibrosis leading to the occurrence and maintenance of atrial fibrillation.
When a large number of myocardial cells are suddenly damaged after myocardial infarction, resulting in the formation of collagen scars (30). Necrotic cells released dangerous signals, which activate innate immune pathways, and trigger a strong inflammatory response. Downstream signals focus on the activation of mitogen-activated protease (MAPK) and NF-κB. These pathways drive the expression of pro-inflammatory genes including inflammatory factors (such as TNF-α, IL-1β, IL-6 and IL-18) (31–33). Inflammation signals promote the adhesion between leukocytes and endothelial cells, leading to extravasation of neutrophils and monocytes. When the infiltrating white blood cells clear the necrotic cells, mediators that inhibit inflammation are released (34, 35). The inhibition of inflammatory response is related to the activation of repair cells, leading to the proliferation of fibroblasts to maintain the integrity of the infarcted ventricle (36–38). TNF-α is an important factor in vascular inflammation, and its level is elevated in vascular diseases. Many effects of TNF-a are similar to Ang II. Arenas et al. report that Ang II regulates endothelial cells secreting inflammatory cytokines TNF-α and matrix metalloproteinase-2 (MMP) (39). Ang II stimulates the production of TNF-α through a PKC-dependent pathway in macrophages (40). In monocytes, macrophages, vascular smooth muscle cells and endothelial cells, TNF-α activates NF-κB, thereby inducing the production of adhesion molecules and chemokines, such as IL-6 and IL-8(41). Cytokines also play an important role in the occurrence and development of atherosclerotic lesions (42). The level of IL-18 expressed in atherosclerotic lesions is elevated. Sahar et al. prove that IL-18 activates Src, PKC, and MAPK. In Ang II stimulated smooth muscle cells, IL-18 is enhanced by activating NF-κB, and Ang II also induces IL-18 receptor mRNA expression through STAT3 (43). Nami K et al. use Ang II to stimulate the HL-1 cell line. Ang II induces reactive oxygen species (ROS) production and activates MAPK, TGF-β1, IL-6, IL-1β, NF-κB, and TNF-α. Ang II regulates atrial fibrillation through inflammatory mechanisms and MAPK signaling pathways produced ROS (44༌45). In addition, TNF-α, IL-6, and IL-1β are also the prototype stress activators of P38-a. Cytokines bind to different types of surface receptors to determine the p38-a phosphorylation pathway, usually with TRAF ubiquitin ligase and TAK1 and others with MAPK3. The P38-a-MK2 pathway regulates the expression of TNF-α and mediated the production of TNF-α induced pro-inflammatory factors, while limites TNF-α induced apoptosis .
This study confirmed that the administration of inflammatory stimuli (TNF-α) has an effect through the MKK-P38-MAPKs signaling pathway. The expression of inflammatory factors was as follows: TGF-β and TNF-α were significantly increased and statistically significant, and IL-6 level was also increased, but there was no significant difference.
The formation and degradation mechanism of angiotensin II (Ang II) is an important factor that determines its ultimate physiological effect. Ang II is an octapeptide. Angiotensin is cut into angiotensin I by aspartase renin, and angiotensin I is converted into Ang II by angiotensin-converting enzyme (ACE) (46). A recently study discovers carboxypeptidase ACE2 cleaves an amino acid from Ang I or Ang II, reduces the level of Ang II and increases the vasodilatory metabolite Ang 1−7 (47). ACE2/Ang1−7/MAS axis regulation regulates fiber generation and remodeling. In another study, male rats are divided into sham operation group, Ang II group, Ang II + Ang 1−7 group, Ang II + Ang 1−7 + A77 group, stimulate for 4 weeks, and finally tissues are collected. Results indicate that chronic Ang 1−7 prevents cardiomyocyte hypertrophy and interstitial fibrosis induced by hypertension. Ang 1−7 acts directly on the heart tissue. It is also confirmed that the anti-fibrosis and anti-hypertrophy effects of Ang 1−7 are not mediated by changes in the number of AT1 or AT2 cardiac receptors (48). In a mouse model of asthma, it is also confirmed that Ang 1−7 inhibits ovalbumin-induced airway leukocyte influx, perivascular and peribronchial inflammation, fibrosis, and goblet cell hyperplasia or metaplasia (49). The ACE2/Ang1−7/MAS axis regulates the recruitment and activation of leukocytes. In the model of pulmonary hypertension, the activation of the ACE2/Ang1−7/MAS axis also regulates the expression of pro-inflammatory factors, reducing the expression of TNF-α, TGF-β, IL-6, IL-1β and increases the expression of anti-inflammatory factor IL-10 (50, 51). Jun Mori implants micro-osmotic pumps in male db/db mice (diabetic cardiomyopathy mice) at the age of 5 months, and gives them Ang 1−7 for 28 days. It is found that Ang 1−7 inhibits the increase of myocardial protein kinase C level and the loss of extracellular signal-regulated kinase 1/2 phosphorylation, and reduces the levels of triglyceride and ceramide in the heart of db/db mice, and increases the expression of triglyceride lipase in myocardial fat (52).
In this study, the protein expressions of MKK3, MKK4, MKK6, PMMK4, and PP38 were significantly increased after inflammatory factor (TNF-α) stimulation, and after intervention with Ang 1−7, the protein expression of MKK3, MKK4, MKK6, PMMK4, and PP38 was significantly lower than the stimulation group (P < 0.05). In terms of inflammatory factors, TGF-β and TNF-α were significantly increased after TNF-α stimulation, and the expression of TGF-β and TNF-α was significantly reduced after Ang 1−7 intervention (P < 0.05). IL-6 also had the same effects as TGF-β and TNF-α trend, but there was no statistically significant.