ROCK inhibitor Y-27632 down-regulates Il-1β expression in mice with experimental autoimmune myocarditis

doi:10.21203/rs.3.rs-3324490/v1

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ROCK inhibitor Y-27632 down-regulates Il-1β expression in mice with experimental autoimmune myocarditis

https://doi.org/10.21203/rs.3.rs-3324490/v1

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published 29 Apr, 2024

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Autoimmune myocarditis is a limited or diffuse inflammation of the myocardium brought on by dysfunction in the normal function of cellular and humoral immunity of the body. Mouse models of experimental autoimmune myocarditis (EAM) were constructed using peptide MyHC-α614–629. After secondary immunization, the mice were given intraperitoneal injection of the Rho kinase (ROCK) inhibitor Y-27632 the next day, and the heart tissues of the EAM mice were isolated and weighed on day 21. As a result, the hearts of EAM mice were significantly enlarged and whitened; the body weight (BW) of mice in the EAM group increased slowly, and the heart weight (HW) and the ratio of HW/eventual body weight (e-BW) were raised; the inflammatory infiltration and fibrosis of the myocardial tissue were aggravated. But Y-27632 treatment improved the above-mentioned phenotypic or pathological features of EAM mice. Besides, the monocytes in the spleen of EAM mice mounted up, which showed a decline in number after Y-27632 treatment. Mechanistic analysis revealed a significant increase in the expression of Notch1, Hes1, Jag2, Dil1, Tlr2 and Il-1β in the myocardial tissue of the EAM mice. Notably, the expression of IL-1β was consistent with that of Notch1 and Tlr2. After Y-27632 treatment, the expression of key target genes (Notch1, Hes1, Dil1 and Jag2) of the NOTCH signaling pathway and Tlr2 expression were obviously decreased. The ROCK inhibitor Y-27632 exerts a protective effect in EAM mice by down-regulating Il-1β expression. This study aims to provide a reference value for the future treatment of myocarditis in clinical settings.

Experimental autoimmune myocarditis (EAM)

Y-27632

Il-1β

MYCN

NOTCH/TLRs pathway

Myocarditis is a limited or diffuse inflammation of the myocardium caused by infectious or non-infectious agents. The infectious agents mainly include viruses, bacteria, mycoplasma, chlamydia, rickettsia and fungi, etc[1, 2]. Non-infectious myocarditis is mainly classified into isolated myocarditis and autoimmune myocarditis. Patients with myocarditis mainly present with palpitations, arrhythmias, chest tightness, chest pain, dyspnea, heart failure, reduced myocardial systolic and diastolic function, and even sudden death. And myocarditis is the main cause of sudden cardiac death in young people[3, 4]. The autoimmune response is an important pathogenesis of myocardial injury whose incidence is increasing[5]. Fortunately, clinical trials have confirmed that clinical symptoms of myocardiopathy can be relieved by immunosuppressive therapy[6]. Experimental autoimmune myocarditis (EAM) mouse model is the current popular experimental model for studying the immune mechanism of myocarditis[4, 7].

Y-27632, a potent and specific Rho kinase (ROCK) inhibitor, can inhibit both ROCK I and ROCK II[8]. On the one hand, Y-27632 plays an important role in cardiovascular disease and stem cell culture[9, 10]. On the other hand, it is involved in a variety of biological processes including cell proliferation, adhesion, apoptosis, cycle regulation and angiogenesis[11, 12]. ROCKs are aberrantly expressed in a variety of cardiovascular diseases, such as cardiac hypertrophy, atherosclerosis, ischemic heart disease, systemic hypertension and pulmonary hypertension[13, 14].

The NOTCH signaling family consists of four protein paralogs (NOTCH1-4) with ligands of Delta-like 1, 3 and 4 as well as Jagged 1 and 2. The NOTCH signaling is a highly evolutionarily conserved signaling pathway[15]. Toll-like receptors (TLRs) are ancient pattern recognition receptors (PRRs) which play a vital role in intrinsic immunity[16]. To date, 12 TLRs have been identified in mice and 10 TLRs in humans[17]. TLR9 is a member of the TLR family. It has been reported that in coxsackievirus B3- (CVB3-) induced viral myocarditis (VMC), the TLR9 signaling pathway promotes cardiac inflammation by activating interferon regulatory factor 5 (IRF5). After interfering with the TLR9-IRF5 signaling pathway, the damage of VMC cardiomyocytes is attenuated[18]. Moreover, TLR4 and nuclear factor kappa B (NF-κB) were hyperactivated in VMC rats. In this case, inhibition of TLR4 and NF-κB expression can reduce apoptosis in VMC rat cardiomyocytes, thereby relieving the disease symptoms. The expression of TLR4 and phosphorylated NF-κB p65 significantly increased in CVB3-induced mouse myocardial tissue, as well as the expression levels of interleukine (IL)-1β, IL-6, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (INF-γ)[19].

Patients with myocarditis typically exhibit a variety of symptoms, including nonspecific chest pain, chest tightness, dyspnea and palpitations[20]. These symptoms are sometimes disregarded due to their resemblance to those of conditions such as coronary artery disease[21]. Therefore, it is particularly important to find effective and specific methods for prevention and treatment of myocarditis. With this goal, this study explored the role and mechanism of the ROCK inhibitor Y-27632 in the treatment of myocarditis through the EAM mouse models.

Establishment of mouse models of experimental autoimmune myocarditis

In the two immunizations on days 1 and 8, emulsions of 0.2 mg of peptide (MyHC-α614–629) were injected subcutaneously at multiple points in the groin of mice, and the mice were dissected on day 21 (Fig. 1A). It was found that the hearts of mice in the EAM group were slightly whitened and significantly enlarged (Fig. 1B). The starting body weight (s-BW) of each mouse and the heart and body weight after dissection were weighed on day 21. As shown in Supplementary Table S1, the heart weight (HW) of the mice in the EAM group was much higher and their BW lower than those of the mice in the control group (Fig. 1C and Fig. 1D). Additionally, the ratio of HW to eventual body weight (e-BW) was significantly elevated in the EAM group compared with the control group (Fig. 1E, Table S1). The above results tentatively demonstrated that the EAM mouse models were successfully established.

Y-27632 treatment ameliorates the symptoms of mice with experimental autoimmune myocarditis

Compared with the saline group, the HW of the Y-27632 2HCl group was obviously reduced and the BW raised, and the differences were statistically significant (Fig. 1C and Fig. 1D, Table S2). As for the ratio of HW/BW after autopsy, it was much lower in mice of the Y-27632 2HCl group than the saline group (Fig. 1E, Table S2). In order to better observe the fibrosis of myocardial tissue from the histological level, Masson staining was performed on the mouse sections. In addition, the whitened hearts of the mice in the Y-27632 2HCl group were improved and the heart volume was significantly reduced (Fig. 2A). The results showed that the EAM group displayed a notable rise in the percentage of blue area compared with the control group, indicating a significant increase in myocardial fibrosis in EMA mice; the Y-27632 2HCl group showed an obvious drop in the percentage of blue area compared with the saline group, indicating that myocardial fibrosis was alleviated by Y-27632 in mice (Fig. 2B and 2C). These experimental results confirmed that Y-27632 2HCl intervention could significantly improve the symptoms in EAM mice.

Y-27632 treatment improves inflammation in mice with experimental autoimmune myocarditis

To detect myocardial inflammation in mice, HE staining and mononuclear cell culture were performed. The HE staining results confirmed that the inflammation score of myocardial tissue was quite higher in mice of the EAM group compared to that of the control group, indicating a significant increase in inflammatory infiltration in EMA mice. After Y-27632 2HCl treatment, the inflammation score was significantly decreased, suggesting that inflammatory infiltration was alleviated by Y-27632 with statistical significance (Fig. 3A and 3B). The spleen is the largest peripheral immune organ. Mononuclear cells contain lymphocytes and monocytes, and the proportion or number of mononuclear cells can indirectly and effectively reflect the inflammatory condition of the organism. Therefore, the mononuclear cell number in mouse spleen can effectively reflect the condition of mice. The experimental results showed that the number of mononuclear cells in the spleen of EAM mice was considerably increased compared with that of the control group, which was decreased markedly after Y-27632 2HCl treatment, implying that the inflammation in the myocardial tissue of mice was relieved (Fig. 3C).

Y-27632 significantly decreases the expression of pro-inflammatory factor Il-1β

The expression of Il-1β, Nos2, Agr1 and TLRs was detected using qRT-PCR. The results showed that both Il-1β and Nos2 expression were elevated in mice of the EAM group compared with those of the control group, but no statistical difference existed in the expression of Nos2. After Y-27632 2HCl treatment, the mice exhibited a decline in Il-1β and Nos2 expression compared with the mice in the saline group, and only Il-1β expression showed statistically different (Fig. 4A-4C). TLRs signaling pathway was associated with inflammation, and the expression of its related genes Tlr2 and Tlr4 was detected by qRT-PCR. In terms of the qRT-PCR results, only Tlr2 expression was significantly up-regulated in mice of the EAM group compared to the control group, which was reduced after Y-27632 2HCl treatment with statistically significant difference (Fig. 4D and 4E). It is logical to speculate that the improvement of symptoms in EAM mice by Y-27632 2HCl may be achieved by the decreased expression of pro-inflammatory factor Il-1β and the inhibition of TLRs activity.

Inhibition of rock activity is beneficial to alleviate the up-regulation of Il-1β caused by notch signaling pathway activation

Y-27632 2HCl treatment was revealed to reduce the expression of inflammatory factor Il-1β in EAM mice. In the investigation of the mechanism of the reduced expression of Il-1β, some researchers have already reported that the expression of Il-1β was associated with NOTCH signaling pathway activity. In addition to this, the above results suggest that it may also be associated with the inhibition of TLRs activity. To confirm these results, western blotting was carried out to examine the expression of Notch1, Il-1β and Hes1. The results showed that the expression of Notch1, Il-1β and Hes1 were all increased in the myocardial tissue of EAM mice, and all of them were significantly decreased after treatment with Y-27632 2HCl (Fig. 5A). As for the Immunohistochemical results, the expression of Il-1β, Hes1 and Tlr2 were all also increased in the myocardial tissues of EAM mice and were all significantly reduced after Y-27632 2HCl treatment (Fig. 5B-G). To further confirm the results, the target genes of the NOTCH signaling pathway, Notch1, Hes1, Dil1 and Jag2, were examined by qRT-PCR. We discovered that the expression of all the target genes was significantly up-regulated in the myocardial tissue of EAM mice, which was down-regulated after Y-27632 2HCl treatment (Fig. 5H-K). Taken together, Y-27632 2HCl treatment of EAM mice reduces the expression of inflammatory factor Il-1β (Fig. 6).

Myocarditis is an inflammatory heart disease that is classified into two categories: infectious and non-infectious[2]. Among them, non-infectious myocarditis is induced by toxic substances, drugs (e.g., immune checkpoint inhibitors), systemic immune diseases, and other factors. This investigation only focuses on autoimmune myocarditis.

Y-27632, the most common ROCK inhibitor, is frequently utilized in studies on cardiovascular disease[22, 23]. ROCK plays an important role in various biological processes such as cell proliferation, metastasis, apoptosis and cycle regulation[24]. The EAM mouse model is currently the most commonly used experimental model for determining the immune mechanism of myocarditis pathogenesis. In this study, we investigated the role of Y-27632 in the treatment of EAM mice. As a result, we discovered that EAM mice had lower BW, higher HW, and higher HW/BW ratios compared to the control group. Further investigation also revealed that the inflammatory infiltration and fibrosis were aggravated in the myocardial tissue of EAM mice, and that there was a considerable rise in the number of mononuclear cells in the spleen. After Y-27632 treatment, BW increased, HW and HW/BW decreased, inflammatory infiltration and fibrosis reduced in myocardial tissues, and the number of mononuclear cells also obviously decreased in the spleen. The results confirmed the role of Y-27632 in the treatment of EAM mice.

The aforementioned outcomes demonstrated that Y-27632 could lessen the symptoms in EAM mice. The expression of Il-1β, Nos2, Agr1 and TLRs was investigated in order to learn more about the mechanism through which Y-27632 works. The expression of Il-1β, Nos2 and Tlr2 was observed to be higher in the myocardial tissue of EAM mice but to be lower after treatment with Y-27632. Notably, the expression of Nos2 showed no statistical difference, so the focus was on Il-1β and Tlr2 expression. Abnormal expression of Il-1β has been reported to be related to the activity of NOTCH/TRLs signaling pathway, and this signaling pathway is crucial in the immune system[25, 26]. The results of the present study showed a consistent expression of Il-1β, Tlr2 and Notch1 in myocardial tissues of mice. In addition, the expression changes of NOTCH signaling pathway target genes Notch1, Hes1, Dil1 and Jag2 were also observed. It follows that Y-27632 ameliorates the symptoms of EAM mice by suppressing the expression of Il-1β.

The ROCK inhibitor Fasudil has been found to play an important role in the treatment of myocarditis in the research of our group. Accordingly, inhibition of ROCK is beneficial to reduce the symptoms of myocarditis. This finding offers a particular reference point for the clinical management of myocarditis in the future. It is also believed that ROCK inhibitors will become more significant in the field of medicine.

Ethics approval

All animal studies were conducted under the protocol approved by the Animal Care and Use Committee of Shanxi Datong University Biomedical research Ethics Review Committee.

Study subjects

Forty Balb/c male mice aged 5–6 weeks were purchased from Beijing HFK Bio-Technology Co., Ltd. The animals were housed in controlled conditions of temperature (range, 20–22°C) and humidity (range, 50–55%) environment, on a reversed 12 h light/dark cycle and with ad libitum access to food and water. Animal maintenance, treatments, and experimental procedures were conducted according to Medical and Biological Ethics Committee regulations, and approved by the Medical and Biological Ethics Committee of Shanxi Datong University.

Mouse model construction

The animals were housed in controlled conditions of temperature (range, 20–22°C) and humidity (range, 50–55%) environment, on a reversed 12 h light/dark cycle and with ad libitum access to food and water. Animal maintenance, treatments, and experimental procedures were conducted according to Medical and Biological Ethics Committee regulations. The 40 Balb/c mice were randomly divided into control group, EAM group, saline group and Y-27632 2HCl group. For the control group, 200 µl of adjuvant dilution (adjuvant: saline = 1:1) was injected subinguinally on days 1 and 8; for the EAM, saline and Y-27632 2HCl groups, two immunizations were performed on days 1 and 8, with 200 µl of emulsions containing 0.2 mg of peptide (MyHC-α614–629) being injected subinguinally in each mouse each time. Specifically, 200 µl of saline was injected through the intraperitoneal cavity every other day starting from day 8 until day 21 in the saline group. As for the Y-27632 2HCl group, 10 mg/(kg·d) of Y-27632 2HCl was injected through the intraperitoneal cavity every other day starting from day 8 until day 21.

Mononuclear cell culture

Firstly, the whole mouse spleen was fully ground using grinder, and then washed with PBS for 5–8 times. After being filtered by cell strainer, the cells were centrifuged at 1000rpm/min for 5min. Finally, 10ml DMEM cell culture medium was added and they were transferred to a 10cm culture dish and cultured at 37 ℃ with 5% CO₂ for 5 days. Following 5 days, the number of mononuclear cell cultured from each mouse spleen was counted, and graphs were made to compare the mononuclear cell number in the control group, the EAM group, the saline group and the Y-27632 2HCl group.

HE staining and immunohistochemistry

HE staining: The specific steps were detailed in the references. Four random photographs were taken under the microscope of each paraffin section, and two pathologists score the inflammation of mice through the double-blind method. The scores were divided into 5 levels, with inflammatory infiltrates of 0–19%, 20%-39%, 40%-59%, 60%-79% and 80%-100% recorded as 0, 1, 2, 3 and 4, respectively. At last, graphs were made for comparative analysis. Hes1, Tlr2, Il-1β and secondary antibodies (rabbit and mouse antibodies) were purchased from Shandong Huaan Biotechnology Co.,Ltd.

Immunohistochemistry: The details of the steps were described in the references. Sections of each mouse were randomly photographed 4 times, and the proportion of brown areas was analyzed using Image statistical software.

Masson staining

Paraffin sections were subjected to dewaxing and hydration. The they were stained with Weigert’s iron hematoxylin for 7 min, Masson’s bluing reagent for 5 min, washed in distilled water for 1 min, phosphomolybdic acid for 2 min, aniline blue solution for 2 min, and dehydrated and decolorized with alcohol for 3 min. After that, the sections were cleared in xylene and mounted in neutral balsa, and finally photographed by microscope. The sections of each mouse were photographed randomly for 4 pictures, and the proportion of blue areas was analyzed using Image statistical software.

Western blotting

Trizol or RIPA was used to extract the total protein. Totally 40 µg of the extracted protein was denatured for western blotting, and the specific experimental procedures were showed in the references[27]. The used antibodies are as follows: Notch1 (rabbit, 1:1000), GAPDH (rabbit, 1:3000), Hes1 (rabbit, 1:500), Il-1β (mouse, 1:1000), and secondary antibody (rabbit or mouse, 1:5000). Notch1 and GAPDH were purchased from CST, and Hes1, Il-1β and secondary antibodies (rabbit and mouse antibodies) from Shandong Huaan Biotechnology Co.,Ltd.

RNA extraction and quantitative real-time PCR

Myocardial tissue RNA was extracted by Trizol method, and the reverse transcription and quantitative PCR steps were referred to the literature[28, 29]. The housekeeping gene Gapdh was selected as the internal control, and the expression differences of target genes between different samples were calculated based on 2^−∆∆Ct method. The primer sequences were listed in Supplementary Table S3.

Statistical analysis

The data were imported into GraphPad Prism 5 as Mean ± standard error of mean (SEM) or standard deviation of mean (SDM). The t-test was used to calculate the P-value for the comparison of two groups, and P < 0.05 indicates statistical difference. Not significant (ns): P > 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Fundings

This study was supported by grants from Basic Research Program Youth Project of Shanxi Province, China (20210302123338) to Y.L., Scientific and Technologial Innovation Program of Higher Education Institutions in Shanxi, China (2021L372) to Y.L. and (2020L0468) to Y.M., College students’ innovative entrepreneurial project in Shanxi Province, China (20230790) to B.X., Innovation and Entrepreneurship Training Program for College Students of Shanxi Datong University, China (2022208) to B.X., and Industry-university-research Project of Shanxi Datong University, China (2022CXY019) to Y.L..

Competing Interests

The authors declare no potential conflicts of interest.

Data Availability

The datasets used during the current study are available from the authors on reasonable request.

Ethics approval

All animal studies were conducted under the protocol approved by the Animal Care and Use Committee of Shanxi Datong University Biomedical research Ethics Review Committee.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Authors' contributions

YL, GG and YH designed the experiments. YL, BX, LS, XY and YL performed the experiments. YL, YM, NZ and CN analyzed and curated the data. YL, CN and YW prepared the manuscript.

Acknowledgments

This study was supported by grants from Basic Research Program Youth Project of Shanxi Province, China (20210302123338) to Y.L., Scientific and Technologial Innovation Program of Higher Education Institutions in Shanxi, China (2021L372) to Y.L. and (2020L0468) to Y.M., Innovation and Entrepreneurship Training Program for College Students of Shanxi Datong University, China (2022208) to N.Z., and Industry-university-research Project of Shanxi Datong University, China (2022CXY019) to Y.L..

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No competing interests reported.

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You are reading this latest preprint version

ROCK inhibitor Y-27632 down-regulates Il-1β expression in mice with experimental autoimmune myocarditis

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Establishment of mouse models of experimental autoimmune myocarditis

Y-27632 treatment ameliorates the symptoms of mice with experimental autoimmune myocarditis

Y-27632 treatment improves inflammation in mice with experimental autoimmune myocarditis

Y-27632 significantly decreases the expression of pro-inflammatory factor Il-1β

Discussion

Materials and methods

Ethics approval

Study subjects

Mouse model construction

Mononuclear cell culture

HE staining and immunohistochemistry

Masson staining

Western blotting

RNA extraction and quantitative real-time PCR

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1