Protection of Ang1-7 througth MKK/P38MAPKs inammatory signal pathway on TNF-α stimulated mouse HL-1 cells

to explore the effect of Ang1-7 througth MKK/P38MAPKs inammatory signaling pathway on TNF-α-stimulated mouse HL-1 cells. Methods Using TNF-α (100 µg/ml) to establish an inammatory atrial brillation model in HL-1 cell, which derived from mouse atrial myocyte. treated HL-1 cells with different concentrations of Ang 1-7 (0.1, 1 and 10 mmol/L) and divided into 5 groups, namely A group(control group), B group(TNF ), C group(TNF + Ang 1-7 0.1 mmol/L), D group(TNF + Ang 1-7 1 mmol/L ) and E group(TNF + Ang 1-7 10 mmol/L ). Firstly, different concentrations of Ang 1-7 (0.1 mmol/L, 1 mmol/L and 10 mmol/L) were used to stimulate for half an hour, and then TNF-α (100 µg/ml) was added to stimulate for four hours. Both the cells and supernatant were collected. Cells were collected for Western Blotting to detect the protein expression of MKK3, MKK4, MKK6, PMMK4 and PP38. The supernatant was subjected to ow cytometry for detecting multi-inammatory factors.


Introduction
Atrial brillation is the most common arrhythmia, its molecular mechanism is unclear. So far, the pathophysiology mechanisms of atrial brillation are not fully understood, the targeted therapy for atrial brillation fails to meet clinical needs. Many researches have clari ed that atrial remodeling is a prerequisite for atrial brillation (1) . Other researches show that atrial brosis is not only related to the stimulation of cardiomyocytes and broblasts, but also related to the activation state of white blood cells. With the release of reactive oxygen species, cytokines and growth factors, white blood cells are recruited and subsequent increased matrix deposition, leading to unfavorable atrial remodeling (2,3) .
These in ammatory pathways are prerequisites for atrial brillation. The angiotensin-aldosteronesystem is an important hormone system in the occurrence and development of atrial brillation.
Therefore, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers are becoming new drugs to prevent atrial brillation (4) .Ang1-7 is produced by angiotensin-converting enzyme 2 (ACE2) catalyzed angiotensin I (Ang I) or angiotensin II (Ang II). The intracellular signal mechanism transmitted by MAS is still unclear. such as Akt phosphorylation, protein kinase C activation and mitogen-activated protein kinase (MAP) inhibition seem not be involved in this signal transduction pathway (5) . Wang et al.
also con rm that Ang1-7 attenuates the expression of heat shock protein (HSP27), thereby inhibiting the occurrence of atrial brillation (6) . the Ang1-7/MAS conduction pathway shoud be further study and makes it an important target for the treatment of Atrial brillation .

Cell culture and groups divided
All cell experiments required ultraviolet rays to irradiate the operating table and the items needed for the experiment for 20 -30 minutes before subsequent experiments carried out. HL-1 atrial myocytes were cultured in DMEM high glucose medium containing 10% fetal bovine serum or DMEM high glucose medium with penicillin/streptomycin (fetal bovine serum: penicillin/streptomycin: DMEM high glucose medium 10: 1.1: 100), in 5% CO2 incubator at 37°C. Usually we used 12-well plates for Western Blotting and ow cytometry for detecting multi-in ammatory factors. Marked the blank control group as A group and A' group, experimental group TNF-α (TNF-α (100 µg/ml)) as B group, B' group, TNF-α + Ang 1-7 (0.1 mm) as C group, C' Group, TNF-α + Ang 1-7(1 mm) as D group, D' group, TNF-α + Ang 1-7 (10 mm) as E group, E' group. Group A and Group A' were repeated controls for each other. The HL-1 cells were given different concentrations of Ang 1-7 (0.1 , 1 and 10 mmol/L) to stimulate for half an hour, and added TNFα (100 µg/ml) to stimulate for 4 h, in 5% CO2 incubator at 37°C. After time was up, the cells and supernatant were collected, the cells were collected for Western Blotting, the supernatant was subjected to ow cytometry for detecting multi-in ammatory factors.

Western Blotting
Different treatments of HL-1 cells were used to extract protein from cell lysates using RAPI. Loaded 10 μl protein samples and subjected to 10% SDS-polyacrylamide gel electrophoresis (140 MV) to separate proteins of different sizes and molecular weights. Transfered the protein to PVDF membrane, 200 MA, 1.5 h. PVDF membrane were blocked with Tris-buffered saline containing 10% nonfat milk at room temperature for 1 h, then incubated with the primary antibodies diluted in TBS at room temperature over night. Primary antibodies used in this study were as followed: anti-P38MAPK antibody (#8690s, 1: 1000), anti-Phospho-p38mapk (Thr180/Tyr182) antibody (#4511, 1 1000), anti-β-actin antibody (1: 1000), anti-mkk3 antibody (1: 1000), anti-phospho-SEK1/mkk4 (C36C11, 1: 1000), Phospho-MKK4c36c11 (1: 1000), MKK6 (Cell signaling technology, 1: 1000) anti-MASsc-390453 (1: 500). Membranes were incubated with the secondary antibody at room temperature for 1 h and visualized using the chemiluminescence reagent ECL advance. TanonGis system was used to perform band gray scale analysis to calculate the net optical density, and the ratio of the net optical density of the target protein to the net optical density of β-actin represented the relative level of the target protein.

Flow cytometry
We chose 10 supernatant EP tubes (respectively con, TNF-α, TNF-α + Ang 1-7 (0.1 mm), TNF-α + Ang 1-7 (1 mm), TNF-α + Ang 1-7 (10 mm), repeated control for each group A and A'). Added beads mouse 10 µl + Mouse Macrophage/Microglla Panel Detection Antibodies 10 µl + legend plex Assay Buffer 20µl to each EP tube and mixed. Then added 10 µl of supernatant to each EP tube and mixed, avoided light and shaked on a vortex shaker at a speed of 3 for 1.5 h. When the time was up, PE 10µl + legend plex Assay Buffer 10 µl were added to each EP tube, and continued to avoid light and shaked on the vortex oscillator at a speed of 3 for 0.5 h. Taken a 15 ml centrifuge tube and added 14.5 ml deionized water (ddH 2 O) + wash Buffer (20×) 750 µl into it. After the shaking was over, added 500 µl of the liquid in the prepared 15 ml centrifuge tube, and centrifuged of 600 rcf for 8 min at 4℃. After centrifugation, blue crystal precipitation could be seen. The upper layer of liquid was removed using a micropipette, leaving blue crystals. 200 µl of the liquid was added in the prepared 15 ml centrifuge tube and subjected to the ow cytometry for multi-factor detection.

Statistical analysis
The results were compared with statistics using SPSS 25.0 software. Measurement data were expressed as mean ± standard deviation (x±s). Signi cance test was performed using one-way analysis of variance (ANOVA), and LSD was used for comparison between groups when the test for homogeneity of variance was equal. When the homogeneity of variance is unequal, the comparison between the groups uses the Tamheni method. P < 0.05 was considered statistically signi cant.

Relative protein expression of MKK3/4/6 and PMKK4
Compared with the A group, the protein expressions of MKK3, MKK4, MKK6, and PMMK4 was signi cantly increased after stimulation with in ammatory factors (TNF-α), which was statistically signi cant (P < 0.05). After intervention with Ang 1-7, the protein expression of MKK3, MKK4, MKK6, and PMMK4 was signi cantly lower than that of the stimulation group (P < 0.05). The protein expressions of MKK4, MKK6 and PMMK4 between groups B, C, and D, E showed an increasing trend, but there was no signi cant difference (P > 0.05). There is no signi cant difference in multiple comparisons of MKK3, MKK4, MKK6, PMMK4 between groups B, C, and D,E (P > 0.05) ( Table 1 and Fig. 1).

Relative Protein Expression Of P38mapk And Pp38mapk
Compared with A group, there was no signi cant difference in P38 protein expression after in ammatory factor (TNF-α) stimulation, but PP38 protein expression was signi cantly increased (P < 0.05). After the intervention of Ang 1-7, the protein expression of PP38 was signi cantly lower than that of A group (P < 0.05), but there was no signi cant difference in the reduction of the protein in group B. There was no signi cant difference in the protein expression of P38. The expression of PP38 protein in groups B, C, and D, E had a decreasing trend, but there was no signi cant difference (P > 0.05). There was signi cant differences of the protein expression of PP38 within multiple comparisons between group A and groups C and D (P < 0.05), and there was signi cant differences between group B and group D (P < 0.05). There was no signi cant difference of the protein expression of PP38 between group B and groups A and C ( Table 2 and Fig. 2).

Expression Of Mas Receptor
Compared with A group, there was no signi cant difference in the protein expression of MAS after stimulation with in ammatory factor (TNF-α). After intervention with Ang 1-7, the protein expression of MAS was higher than that of B group and C group, but there was no signi cant difference (P > 0.05). The expression of MAS protein in groups C, D, and E showed an increasing trend, but there was no signi cant difference (P > 0.05). There was no signi cant difference of MAS protein expression in multiple comparisons within the groups (P > 0.05) ( Table 3 and Fig. 3). 2.4 Flow multi-factor detection of the expression of in ammatory factors of TGF-β, IL-6, TNF-α Compared with the A group, the TGF-β of group B was signi cantly increased after stimulating factor (TNF-α) was given, and it was statistically signi cant (P < 0.05). The expression of TGF-β was decreased after the intervention of Ang 1-7, and there was a signi cant difference in group E (P < 0.05). There is no signi cant difference between group C, D and group B (P > 0.05). There is no signi cant difference in multiple comparisons within the group between C,D and E(P > 0.05) ( Table 4 and Fig. 4). Compared with A group, IL-6 in group B increased after stimulating factor (TNF-α) was given. The expression of TGF-β decreased after the intervention of Ang 1-7,(P > 0.05) ( Table 4 and Fig. 4). Compared with the A group, TNF-α in group B was signi cantly increased after stimulating factor (TNF-α) was given, and it was statistically signi cant (P < 0.05). The expression of TNF-α was signi cantly decreased after the intervention of Ang 1-7, and it was statistically signi cant (P < 0.05). However, there was no signi cant difference in multiple comparisons between groups C, D and E (P > 0.05) ( Table 4 and Fig. 4).

Discussion
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, in ammation, 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 in ammation, immune disorders or cancer (8) . The activation of P38 is mediated by the phosphorylation of speci c 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, brosis and apoptosis (10,11 12) .
Atrial muscle brosis leads to local conduction slowdown and conduction disorders, leading to unidirectional conduction block (13) . Fibrosis increases the number of broblasts and change their characteristics, by changing the interaction between cardiomyocytes and broblasts to couple the electrophysiological behavior of cardiomyocytes, thereby promoting atrial brillation (14) . The role of the RAAS system in the remodeling of atrial structure has been con rmed. Clinical studies have shown that RAAS inhibitors are bene t for patients with atrial brillation after pulmonary vein isolation (15) . RAAS inhibitors also inhibit atrial brosis and atrial remodeling, and delay atrial brillation (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 in ammatory collagen ber deposition and causing atrial brosis. In animals with an overexpression of angiotensin converting enzyme, Ang II levels are signi cantly increased, and there was obvious atrial enlargement, atrial brosis and atrial brillation. 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 brosis. In a clinical study, 56 patients with rheumatic heart disease are divided into atrial brillation group and sinus rhythm group. Left atrial appendage tissue is collected during cardiac surgery to assess myocardial brosis. The study found that the atrial MAPKs activity in the atrial brillation group is signi cantly higher than sinus rhythm group, the atrial TGF-β1 and CTGF mRNA and protein expression in patients with atrial brillation increases signi cantly, con rming that the MAPKs/TGF-β1/TRAF6 signaling pathway is involved in the occurrence of atrial brosis in patients with atrial brillation (21 22) . In addition, the increased expression of TGF-β1 induced by Ang II is the main mechanism of Ang II-induced myocardial brosis (23) . Ang II increased the activation of mouse broblast MAPKs, up-regulate the expression of TGF-β1 and CTGF, and promote the proliferation of broblasts (24) .
The increase in CTGF expression induced by Ang II or TGF-β1 is an important factor involved in atrial brosis and atrial brillation. In vitro studies on mouse broblasts show that Ang II stimulates TGF-β1 on atrial broblasts, which acts in a Smad-independent manner. In this study, it is also con rmed that the TGF-β1/TRAF6 signaling pathway is involved in atrial brosis.
Transforming growth factor (TGF-β1) is a key broblast growth factor. TGF-β1 regulates cell proliferation, apoptosis and migration, and regulate the synthesis of extracellular matrix (such as up-regulating the expression of bronectin and collagen bers). Overexpression of TGF-β1 may cause atrial brosis and atrial brillation. The increase in CTGF expression induced by Ang II or TGF-β is an important factor involved in atrial brosis and atrial brillation (25) . TGF-β may be the main switch that regulates the transition from in ammatory response to brosis (26) . Masaki Ikeuchi et al. show that early inhibition of TGF-β aggravates ventricular dysfunction and in ammatory response, while late destruction of TGF-β signaling protects interstitial brosis 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 speci c destruction of TGF-β receptors by cardiomyocytes has a protective effect and extensively stimulates anti-in ammatory 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 re ect the loss of anti-in ammatory effects of in ammatory cells, endothelial cells or broblasts (29) . This study con rmed that in ammatory 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 signi cantly increased, and the concentration of in ammatory factors TGFβ also increased. Thus, these changes may cause myocardial brosis leading to the occurrence and maintenance of atrial brillation.
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 in ammatory response. Downstream signals focus on the activation of mitogen-activated protease (MAPK) and NF-κB. These pathways drive the expression of proin ammatory genes including in ammatory factors (such as TNF-α, IL-1β, IL-6 and IL-18) (31)(32)(33) . In ammation signals promote the adhesion between leukocytes and endothelial cells, leading to extravasation of neutrophils and monocytes. When the in ltrating white blood cells clear the necrotic cells, mediators that inhibit in ammation are released (34,35) . The inhibition of in ammatory response is related to the activation of repair cells, leading to the proliferation of broblasts to maintain the integrity of the infarcted ventricle (36)(37)(38) . TNF-α is an important factor in vascular in ammation, 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 in ammatory 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 brillation through in ammatory 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-in ammatory factors, while limites TNF-α induced apoptosis . This study con rmed that the administration of in ammatory stimuli (TNF-α) has an effect through the MKK-P38-MAPKs signaling pathway. The expression of in ammatory factors was as follows: TGF-β and TNF-α were signi cantly increased and statistically signi cant, and IL-6 level was also increased, but there was no signi cant 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/Ang 1−7 /MAS axis regulation regulates ber 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 + A 77 group, stimulate for 4 weeks, and nally tissues are collected. Results indicate that chronic Ang 1−7 prevents cardiomyocyte hypertrophy and interstitial brosis induced by hypertension. Ang 1−7 acts directly on the heart tissue. It is also con rmed that the anti-brosis 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 con rmed that Ang 1−7 inhibits ovalbumin-induced airway leukocyte in ux, perivascular and peribronchial in ammation, brosis, and goblet cell hyperplasia or metaplasia (49) . The ACE2/Ang 1−7 /MAS axis regulates the recruitment and activation of leukocytes. In the model of pulmonary hypertension, the activation of the ACE2/Ang 1−7 /MAS axis also regulates the expression of pro-in ammatory factors, reducing the expression of TNF-α, TGF-β, IL-6, IL-1β and increases the expression of anti-in ammatory 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 signi cantly increased after in ammatory factor (TNF-α) stimulation, and after intervention with Ang 1−7 , the protein expression of MKK3, MKK4, MKK6, PMMK4, and PP38 was signi cantly lower than the stimulation group (P < 0.05). In terms of in ammatory factors, TGF-β and TNF-α were signi cantly increased after TNF-α stimulation, and the expression of TGF-β and TNF-α was signi cantly 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 signi cant.