Irisin ameliorates myocardial ischemia-reperfusion injury through modulation of gut microbiota and intestinal permeability in rats

doi:10.21203/rs.3.rs-2248784/v1

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Irisin ameliorates myocardial ischemia-reperfusion injury through modulation of gut microbiota and intestinal permeability in rats

https://doi.org/10.21203/rs.3.rs-2248784/v1

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Purpose Irisin has a significant protection the myocardial from ischemia reperfusion (I/R) injury. Recent studies have suggested that the gut microbiota plays an important role in the progression of myocardial I/R injury. In this context, the aim of this study is to investigate whether Irisin interferes in gut microbiota and gut mucosal barrier during myocardial I/R injury.

Methods All the rats received intraperitoneal injection of irisin or PBS, and then myocardial I/R rats disease model was established. Gut microbiota was analyzed by 16S rRNA high-throughput sequencing to assess changes in the microbiota. Colon and ileum tissue structural damage and apoptosis were evaluated by HE staining and Tunnel, respectively. Intestinal barrier integrity was valuated using by immunofluorescence to detecte the protein expression of ZO-1 and occludin and serum lipopolysaccharide. The proinflammatory cytokines (IL-1β, IL-6, TNF-α) in the colon and ileum tissue by Western Blot. Myocardial HE staining and serum cTnI, CK were performed to evaluate the cardiac function.

Results Irisin treatment significantly improved the imbalance of intestinal flora in I/R rats. And irisin maintained intestinal barrier function by increasing the expression of ZO-1 and occludin protein in colon and ileum and and decreasing serum LPS concentration. Meanwhile, intestinal inflammation in I/R rats was attenuated by irisin. More importantly, irisin was protective against I/R injury in vivo.

Conclusion Irisin intervention could improve the impaired gut mucosal barrier and reduce the production of LPS after I/R through regulating gut microbiota, thus inhibiting the inflammation and finally exerted the cardioprotective effect.

Irisin

Myocardial ischemia reperfusion

Gut microbiota

Intestinal permeability

Inflammation

Acute myocardial infarction (AMI) is a common cardiac emergency associated with high rates of morbidity and mortality [1]. For patients with ST elevation myocardial infarction (STEMI), thrombolytic/fibrinolytic therapy or percutaneous coronary intervention (PCI) as the effective reperfusion strategy that should be performed [2]. Since reperfusion can induce the excessive production of reactive oxygen species, excessive inflammatory response and cell apoptosis, the process is known as myocardial I/R injury [3]. Considering that, avoiding the occurrence of I/R is the key to the treatment of ischemic heart disease. However, there is still no effective therapy and potential target to prevent myocardial I/R injury in patients.

Although the I/R mechanism is not yet cleared, an increasing number of studies have shown that the gut microbiota seems to play an important role in the development [4–6]. An animal studies have suggested that vancomycin decreased susceptibility of the heart to injury in an in vivo model of regional myocardial I/R by reducing the abundance of gut microbiota [7]. In patients with STEMI, the systolic function of the heart decreases, resulting in insufficient blood supply to systemic organs including the intestine, leading to dysbiosis and changes in intestinal permeability [8]. In the meanwhile, the increase in intestinal permeability leads to the translocation of bacterial endotoxins into the blood, which obviously contributes to systemic inflammatory response [9]. Here, we argue that changes in gut microbiota and intestinal permeability are related to the occurrence of adverse cardiovascular events after myocardial I/R injury.

Irisin is a circulating hormone, which is cleaved from the precursor protein fibronectin type III domain containing 5 (FNDC5) [10]. An increasing proportion of evidence has shown that irisin has beneficial effects on cardiovascular diseases [11–13]. Meanwhile, our previous studies have also confirmed that irisin treatment modulated the mitochondrial function via the AMPK pathway, and ultimately protecting the H2C9 cardiomyocytes from hypoxia and reoxygenation injury [14]. In addition, it could not be ignored that irisin had potential to alleviate intestinal inflammation via altering the gut microbiota [15, 16, 17]. Besides, irisin could also restore gut barrier function via the integrin αVβ5-AMPK-UCP 2 pathway [18]. Therefore, this study aims to evaluate the role of the irisin in the rat model of myocardial ischemia-reperfusion injury on intestinal bacteria and intestinal barrier.

2.1 I/R rats model establishment and treatment

Male Sprague Dawley rats (2 months old, 250 ± 20 g) were obtained from the Laboratory Animal Center of Shanxi Provincial People's Hospital. For one week prior to the experiment, the rats were housed in a specific pathogen-free (SPF) facility at a constant room temperature. They had free access to standard mouse chow and tap water. All rats were randomly divided into 3 groups including the sham-operated group (Sham, n = 11), myocardial ischemia reperfusion injury group (I/R, n = 11). Irisin group (Irisin, n = 11). The rats in the Sham and I/R groups were intraperitoneally injected with 0.2 mL PBS. The rats in the Irisin group were intraperitoneally injected with 0.2 mL Irisin (100 mg/kg, Cayman Chemical, USA) [19]. Thirty minutes after the intraperitoneal injection, the I/R model was established as previously described [20]. The rats were intraperitoneally injected with pentobarbitone sodium (50 mg/ kg) and intubated with a small animal ventilator (Shanghai Yuyan Instruments Co, Ltd, China) set at a respiratory rate of 60–70 breaths per minute. The surgical area was disinfected, opened the left chest at the 3rd intercostal space to expose the heart and separated the pericardium to exteriorized the heart. The left anterior descending coronary artery was quickly ligated with 6.0 prolene suturewe for 30 min, and then the suture was removed to be reperfused for 120 min. Meanwhile, the rats had an electrocardiogram (ECG) record (Shanghai Yuyan Instruments Co, Ltd, China) in limb lead II tracing during the operation. Sham-operated rats underwent the same surgical procedures as with the I/R group, except for ligation the left anterior descending coronary artery. The study was permitted by the ethical committee of LinFen Central Hospital (Permit Number: 2021-29-1).

2.2 Serum Cardiac Troponin I, Creatine phosphokinase, and Lipopolysaccharide measurements

After rats were sacrificed, their blood serum was centrifuged. Serum levels of Cardiac Troponin I (cTnI), Creatine phosphokinase (CK) and Lipopolysaccharide (LPS) were determined using the cTnI ELISA kit (Solarbio, China), CK ELISA kit (Solarbio, China), LPS ELISA kit (Signaway Antibody, USA) according to the manufacturer’s instructions.

2.3 Histopathological staining

Colon and ileum tissues were fixed in 4% buffered paraformaldehyde for 48 h and then embedded in paraffin. The sections (5 µm) were mounted on slides, deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and subjected to hematoxylin and eosin (H&E) staining. The histological score was determined as previously described [21–22].

2.4 TUNEL, Immunofluorescence (IF) staining

The sections (5 µm) were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. A TUNEL kit (Beyotime, China) was used for TUNEL staining according to the manufacturer's instructions. Apoptotic rate was counted as described earlier [23]. The sections were submerged into Tris-ethylenediaminetetraacetic acid antigenic retrieval buffer and heated for 5 min at 121°C by autoclave. The sections were then treated with 3% hydrogen peroxide in methanol, blocked with 5% bovine serum albumin, and incubated with antibodies against zonula occludens-1 (ZO-1, Abcam, UK), anated occludin (Abcam, UK) overnight at 4°C, further incubated with Alexa Fluor 59-conjugated secondary antibodies (Bioss, China). Nuclear staining was achieved by 4’ 6- diamidino- 2- phenylindol (DAPI, BOSTER, China). An Olympus inverted fluorescence microscope was used to observe the staining of the sections. The images were evaluated using the Image J software.

2.5 Western blot analysis

Colon and ileum tissues (0.5 g) was lysed on ice with 500 µL of RIPA lysis buffer for 30 min in the presence of protease and phosphatase inhibitors and then sonicated for 1min at 60 Hz. After centrifugation at 12,000 rpm for 15 min at 4°C, the supernatant was harvested. Protein concentrations were determined with the BCA protein assay kit (Solarbio, China) and the protein was thermally denatured at 100°C for 10 min. The protein was then isolated by sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking the membranes with skimmed milk powder, the membranes were incubated overnight at 4°C with rabbit anti-mouse primary antibodies including interleukin-1-beta (IL-1β, CST, USA), interleukin-6 (IL-6, CST, USA), tumour necrosis factor-alpha (TNF-α, CST, USA), and β-actin (CST, USA). After washing with TBST, the membranes were incubated with goat anti-rabbit secondary antibody (CST, USA) at 37°C for 1 h followed by incubation with a chemiluminescent substrate for visual detection using the Tanon imaging system. Image-J software was used to calculate the IODs.

2.6 Extraction of fecal DNA and high-throughput sequencing

DNA in the feces was extracted with the DNA extraction kit, and the quality of DNA extraction was determined by 0.8% agarose gel electrophoresis and quantified by a UV spectrophotometer. The 16S rRNA hypervariable region (V3-V4) was amplified by PCR using the extracted DNA as a template. The primer sequences were ACTGCATCCGCAGCGTCGA and CCTGTACGGTCTTGCATAT. The PCR products were detected with 2% agarose gel electrophoresis and purified with AXYGEN gel extraction kit. The preliminary quantification results of electrophoresis were obtained by fluorescing the PCR amplified products using the Quant-iT PicoGreen dsDNA assay kit. According to the quantitative results, purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego,USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

2.7 Bioinformatic analysis

To minimize the effects of sequencing depth on alpha and beta diversity measure, the number of 16S rRNA gene sequences from each sample were rarefied to 20,000, which still yielded an average Good’s coverage of 99.09%, respectively. Bioinformatic analysis of the gut microbiota was carried out using the Majorbio Cloud platform (https://cloud.majorbio.com). Based on the OTUs information, rarefaction curves and alpha diversity indices including observed OTUs, Chao1 richness, Shannon index and Good’s coverage were calculated with Mothur v1.30.1.The similarity among the microbial communities in different samples was determined by principal component analysis (PCA), principal coordinate analysis (PCOA) and partial least squares discrimination analysis (PLS-DA) based on Bray-curtis dissimilarity using Vegan v2.5-3 package. The PERMANOVA test was used to assess the percentage of variation explained by the treatment along with its statistical significance using Vegan v2.5-3 package. The linear discriminant analysis (LDA) effect size (LEfSe) (http://huttenhower.sph.harvard.edu/LEfSe) was performed to identify the significantly abundant taxa (phylum to genera) of bacteria among the different groups (LDA > 4.00, P < 0.05).

2.8 Statistical Analysis

The data are presented as the mean ± SD of 5 or more independent experiments. Normal distribution was confirmed by the Shapiro-Wilk test. One-way analysis of variance (ANOVA), LSD or Tamhane test were used to compare the statistical differences among multiple groups. A value of p < 0.05 was considered significantly different. SPSS 22.0 were used for statistical analysis.

3.1 Effects of irisin on gut microbial diversity and richness

To characterize the microbial populations in the feces of the rats, we measured bacterial populations by 16S rRNA gene sequencing. In gut microbial α diversity, both richness and evenness were indicated by the Chao index, ACE index, Simpson index and Shannon index. The I/R rats ACE index and Shannon index increased and Simpson index decreased, while irisin completely restored the effect (Fig. 1a).

Principal component analysis (PCA), principal coordinate analysis (PCOA) and Partial least squares discrimination analysis (PLS-DA) were employed to measure the degree of difference between microbial communities. After treatment with irisin, the aggregation of the flora significantly stayed away from the I/R group (Fig. 1b). Notably, the microbial community composition of the rats in the irisin group was similar to the sham group.

Figure 4C was shown that 544 of the total richness of 711 OTUs were shared among all the groups, and there were OTUs between two groups or in each group (Fig. 1c). In addition, irisin treatment decreased OTUs in I/R rats

These data suggested that irisin treatment significantly improved α and β diversity of intestinal microbiota.

3.2 Effects of irisin on the gut microbiota composition

From our research results, it is found that gut microbiota was significantly changed. Then, we compared the gut microbiota composition among the three groups to identify the potentially probiotics or harmful bacteria of irisin intervention after I/R.

At the phylum level, I/R affected the relative abundance of Firmicutes and Actinobacteriota. Interestingly, under the irisin treatment dramatically increased the relative abundance of Firmicutes and decreased the relative abundance of Actinobacteriota (Fig. 2a).

The top 10 the families were significantly affected by the I/R, which was observed that some families (Corynebacteriaceae, Bifidobacteriaceae, Staphylococcaceae, Aerococcaceae, Akkermansiaceae and Carnobacteriaceae) drastically increased, whereas others (Peptostreptococcaceae and Monoglobaceae) decreased. Nevertheless, irisin remarkably increased Peptostreptococcaceae and Monoglobaceae, while decreased Corynebacteriaceae, Bifidobacteriaceae, Staphylococcaceae, Aerococcaceae, Akkermansiaceae and Carnobacteriaceae to some extent (Fig. 2b).

Next, in the top 10 genera were analyzed. In the I/R group, it was observed that some genera (Corynebacterium, norank_f__Erysipelotrichaceae, Bifidobacterium, Staphylococcus, Staphylococcus and Jeotgalicoccus) significantly increased, whereas others (Romboutsia, Turicibacter and Monoglobus) decreased drastically. In contrast, irisin treatment significantly enhanced Romboutsia, Turicibacter and Monoglobus, while remarkably reduced Corynebacterium, norank_f__Erysipelotrichaceae, Bifidobacterium, Staphylococcus, Staphylococcus and Jeotgalicoccus (Fig. 2c).

It was found that the relative abundance of 10 species was different in rats having I/R-induced, with an increase of 7 species (Lactobacillus_johnsonii, uncultured_Allobaculum_sp._g__norank, Bifidobacterium_animalis, Corynebacterium_stationis, uncultured_bacterium_g__Dubosiella, Staphylococcus_lentus_g__Staphylococcus and uncultured_bacterium_g__Jeotgalicoccus) and a decrease of 3 species (Romboutsia_ilealis, uncultured_bacterium_g__Turicibacter and gut_metagenome_g__Lactobacillus). In the Irisin group, an increase of 3 species (Romboutsia_ilealis, uncultured_bacterium_g__Turicibacter and gut_metagenome_g__Lactobacillus) and a decrease of 6 species (uncultured_Allobaculum_sp._g__norank, Bifidobacterium_animalis, Corynebacterium_stationis, uncultured_bacterium_g__Dubosiella, Staphylococcus_lentus_g__Staphylococcus and uncultured_bacterium_g__Jeotgalicoccus) shown (Fig. 2d).

The bacterial composition with remarkable differences among the sham group, the I/R group, and the Irisin group was analyzed by the linear discriminant analysis (LDA) effect-size method (LEfSe) (Fig. 3). g__Turicibacter and s__uncultured_bacterium_g__Turicibacter played critical roles and could be seen as a biomarker in the Sham group. In addition, p__Actinobacteriota, c__Actinobacteria, f__Bifidobacteriaceae, g__Bifidobacterium, o__Bifidobacteriales, s__Bifidobacterium_animalis, g__Dubosiella, s__uncultured_bacterium_g__Dubosiella, s__uncultured_Allobaculum_sp__g__norank, g__norank_f__Erysipelotrichaceae functioned importantly and could be employed as a biomarker in the I/R group, whereas g__Romboutsia, s__Romboutsia_ilealis, f__Peptostreptococcaceae, o__Peptostreptococcales-Tissierellales, p__Firmicutes played acrucial part and could be taken as a biomarker in the Irisin group.

3.3 Irisin maintains intestinal integrity in I/R rats

The gut epithelial integrity is considered as the first line of defense of the gastrointestinal tract. Intestinal dysbiosis in I/R animals may affect gut permeability and subsequently lead to release of bacterial LPS into the blood [24]. In the current study, it was demonstrated that I/R dramatically increased intestinal permeability and damaged the intestinal mucosa (Fig. 4–5), which however were restored by the irisin treatment.

The colon tissue of representative rats of each group showed that the I/R group exhibited obvious inflammatory cell infiltration (Fig. 4a) and apoptosis (Fig. 4b). Meanwhile, it is important to note that in the I/R group, the ileum villi the arrangement was loose, disordered and apoptosis (Fig. 5a-b). After treatment with the irisin, the colon and the ileum structure and the level of apoptosis were alleviated. Inflammatory cell infiltration was decreased in colon tissue (Fig. 4a) and shape of ileum villi was straight finger-like protrusions arranged neatly and densely in the Irisin group (Fig. 5a).

Since intestinal mucosa damage is closely related to the expression of epithelial mucosal proteins and the tight junction proteins, the expression of ZO-1 and occludin was examined. Then, it was revealed that irisin significantly increased ZO-1 and occludin expression compared with the I/R group in both colon and ileum tissues (Fig. 4c, 5c).

These findings indicate that irisin may enhance intestinal barrier integrity in myocardial ischemia-reperfusion injury rats.

3.4 Effects of irisin on gut inflammation

The data obtained confirm that myocardial ischemia-reperfusion dramatically increased intestinal permeability, leading to release of bacterial LPS, which however was restored by the irisin treatment (Fig. 5b). Previous studies revealed that leaky gut would produce higher levels of proinflammatory cytokines in colon or ileum tissues, including IL-1β, IL-6 and TNF-α [25]. Here, in our study, protein expression of these cytokines was measured by Western blot. Then, it was found that IL-1β, IL-6 and TNF-α proteins expression levels were higher in colon and ileum tissues of the I/R rats compared with those of the sham rats (Fig. 5a, 5c). Notably, the protein expression level of these cytokines was altered by irisin treatment, resulting in the protein expression level being close to that of the sham rats (Fig. 5a, 5c).

These results show that irisin reduces inflammation in I/R rats by reducing proinflammatory cytokines in colon and ileumtissues.

3.5 Irisin reduces myocardial injury

Previous studies revealed that ischemia for 30 min would lead to significant elevation of the ST segment of ECG, and the ST segment decreased by at least 50% followed by 120 min reperfusion as a sign of successful model establishment [6]. The myocardial interstitium showed inflammatory cell infiltration and marked edema accompanied by dissolution, rupture and even necrosis of myocardial fibers in I/R rats [26]. In the current study, irisin treatment significantly reduced the elevation of ST segment compared with I/R (Supplementary Figure S1 ). Meanwhile, the infiltration of inflammatory cells and edema was reduced in the myocardial interstitium, and the myocardial fibers were intact and arranged neatly in the rats of irisin group (Supplementary Figure S2a ). Cardiomyocyte necrosis can release a variety of myocardial enzymes to reflect the degree of myocardial injury. As shown in Supplementary Figure S2b, the levels of serum myocardial enzymes increased in I/R group. Notably, the levels of cTnI and CK was altered by irisin treatment, resulting in being close to sham rats.

The data obtained confirm that irisin ameliorates myocardial ischemia-reperfusion injury.

3.6 Gut microbiota-associated blood parameters, colon and ileum barrier function, degree of bacterial translocation and inflammatory response

From heatmap correlation analysis, it is seen that in the 10 top species, 3species were notably associated with blood parameters, colon and ileum barrier function, degree of bacterial translocation and inflammatory response (Fig. 7). uncultured_bacterium_g__Turicibacter and Romboutsia_ilealis were obviously and positively correlated with expression of tight junction protein ZO-1 and occludin in colon and ileum, but noticeably negatively correlated with the levels of cTnI and CK in serum, HE score, apoptosis rate, inflammatory factors (IL-1β, IL-6 and TNF-α) in colon and ileum,and LPS concentration in serum(Fig. 7). Thus, these bacterial species may inhibit I/R development. In addition, uncultured_ Allobaculum_ sp._g__ norank was observably correlated with expression of tight junction protein ZO-1 and occludin in colon and ileum in a negative way, but positively correlated with the levels of cTnI and CK in serum, HE score, apoptosis rate, inflammatory factors (IL-1β, IL-6 and TNF-α) in colon and ileum,and LPS concentration in serum in an obvious manner (Fig. 7). Thus, these bacterial species may induce I/R. All in all, these findings demonstrated that the 3 bacterial species play vital roles in myocardial I/R injury.

Evidence from a compilation of studies of animals and humans indicates that the implications of gut microbiota and their metabolites in cardiovascular diseases are well established [27, 28]. Our previous studies showed that irisin could protect the H2C9 cardiomyocytes from hypoxia and reoxygenation injury [14]. However, if there is a crosstalk between irisin, gut microbiota and cardioprotection remains unclear. In this study, our data indicate that myocardial I/R injury is accompanied by intestinal microbiota imbalance. Interestingly, irisin treatment significantly decreased the abundance of gut microbiota. Besides, the treatment also decreased gut inflammation and maintained the integrity of the intestinal barrier. As far as we have known, this is the first investigation that the effect of irisin on intestinal flora under the myocardial I/R injury stress.

Then, we performed 16S rRNA gene sequencing on the gut microbiota. In this study, significant microbiota changes at both I/R and Irisin group of cecal contents were observed. Our data were compatible with a recent study that I/R rats α diversity significantly increased [29]. β diversity revealed significant separation of the community compositions between the Sham and I/R groups, further confirming the possibly close relationship between myocardial I/R injury and microbiota. It is important to note that the distribution of microbiota is relatively discrete in I/R rats, which may be due to the difference in the degree of acute stress. Moreover, Irisin prevented acute intestinal stress and thus made the microbiota similar to that of the sham group. The possibility was raised by Zhou et al. [30] that the increased richness and distinct structure of microbiome may be caused by the transportion of intestinal bacteria into the blood of I/R rats. In the present study, the relative abundance of gut bacteria changed significantly in the phylum, families, genera and species lever. LEfSe results were employed to analyze the potential pathogenic bacteria in the intestinal of I/R rats, such as p__Actinobacteriota, c__Actinobacteria, f__Bifidobacteriaceae, g__Bifidobacterium, o__Bifidobacteriales, s__Bifidobacterium_animalis, g__Dubosiella, s__uncultured_bacterium_g__Dubosiella, s__uncultured_Allobaculum_sp__g__norank, g__norank_f__Erysipelotrichaceae. It is well known that Bifidobacteriaceae is a common probiotic that maintains gut microbiota balance [31]. Nevertheless, the results in this study are the opposite. If the relative abundance of probiotic is too high, the balance of bacterial flora will may be broken and become potential pathogenic bacteria. Data supporting probiotic role in other conditions are often conflicting [32]. One possible explanation for these results is that the different gut environments may affect the abundance and composition of gut microbiota. Apart from that, g__Romboutsia, s__Romboutsia_ilealis, f__Peptostreptococcaceae, o__Peptostreptococcales-Tissierellales, p__Firmicutes played acrucial part and could be taken as a potential probiotic in the Irisin group. Romboutsia species, such as Romboutsia ilealis [33] and Peptostreptococcaceae [34] were able to utilize glucose and carbohydrates to generate Short chain fatty acids to promote intestinal barrier integrity. Most of Firmicutes are probiotics, except Lactobacillus, other beneficial bacteria can produce butyrate [35]. Importantly, butyrate in particular is important for maintaining health via regulation of the immune system, maintenance of the epithelial barrier [13, 36]. In brief, the findings obtained demonstrate that the improvement of myocardial ischemia-reperfusion injury by irisin is closely related to the changes of intestinal flora.

Myocardial infarction causes ischemic stress, such as intestinal hypoperfusion, loss of tight junction protein occludin and intestinal mucosal damage, and increased intestinal permeability [37]. Tight junctions are important in the intestinal mucosal barrier, and they are located at the top of the intestinal epithelium and consist of a number of proteins which include ZO-1 and occludin of adjacent intestinal cells [38]. In the current study, the structure of the colon and ileum was destroyed, the apoptosis of intestinal epithelial cells was significantly increased and the expression of ZO-1 and occludin proteins was decreased in the I/R rats. These results suggest that myocardial ischemia-reperfusion can increase intestinal permeability in rats, but irisin treatment can reverse this effect. It may be that irisin increases the abundance of beneficial bacteria in the intestine to maintain intestinal barrier integrity. Gut barrier breakdown leads to bacteria and endotoxin (LPS) translocation into the systemic circulation [39]. Then, low levels of LPS in the blood may activate TLR4 signaling in various cells, causing systemic and targeted inflammation [40–40]. In this case, we determined serum LPS concentration and the expression of inflammatory factors proteins in colon and ileum tissues. Our results indicate that irisin inhibited the transfer of LPS from the intestine to the systemic circulation and over-expression the inflammatory factors of IL-1β, IL-6 and TNF-α in I/R rats. Therefore, the beneficial effects induced by irisin treatment may be attributed to specific changes in the gut microbiota and maintenance of intestinal barrier integrity.

Here, this study indicated that intestinal microbiota is involved in irisin alleviating myocardial ischemia-reperfusion injury. The effects of gut microbiota and intestinal barrier are likely to be part of the mechanism of irisin treatment process. However, the pathway through which irisin affects intestinal microbiota and its effects on metabolites still need to be explored in depth.

Myocardial ischemia-reperfusion promotes alterations in gut microbiota that may enhance intestinal permeability and LPS translocation. However, irisin supplementation reversed the changes in gut microbiota, restored intestinal barrier structure, reduced LPS translocation, and decreased inflammatory response, thus finally exerted the cardioprotective effect. In conclusion, the irisin treatment can be an important tool for preventing and treating patients with myocardial ischemia-reperfusion and clarify that intestinal flora is the mechanism affecting myocardial ischemia-reperfusion injury, which can develop new prevention and diagnostic strategy to promote the health of patients with myocardial ischemia-reperfusion.

I/R, ischemia reperfusion; AMI, acute myocardial infarction; STEMI, ST elevation myocardial infarction; PCI, percutaneous coronary intervention; SPF, specific pathogen-free; ECG, electrocardiogram; cTnI, Cardiac Troponin I; CK, Creatine phosphokinase; LPS, lipopolysaccharide; H&E, hematoxylin and eosin; IF, immunofluorescence; ZO-1, zonula occludens-1; DAPI, 4’ 6- diamidino- 2- phenylindol; IL-1β, interleukin-1-beta; IL-6, interleukin-6; TNF-α, tumour necrosis factor-alpha; PCA, Principal component analysis; PCoA, principal coordinate analysis; PLS-DA, partial least squares discrimination analysis, LDA, linear discriminant analysis; LEfSe, linear discriminant analysis effect size; ANOVA, One-way analysis of variance

Authors’ Contributions All authors contributed to the study conception and design. Qingqing Liu, Yu Zhu, Guangyao Li, Tiantian Guo, Mengtong Jin, Duan Xi, Shuai Wang, Xuezhi Liu conducted the research. Data analysis were performed by Qingqing Liu, Yu Zhu, Guangyao Li and Duan Xi. The first draft of the manuscript was written by Qingqing Liu and all authors commented on previous versions of the manuscript. Ronghua Liu, Jiamao Fan had primary responsibility for the final content. All authors read and approved the final manuscript.

Funding This work was supported by the Scientific research project of Shanxi Provincial Health Commission (2022122) and the Linfen City key research and development plan (2108).

Data Availability The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval Animals were housed and cared for in facilities run by the Laboratory Animal Centre and were routinely monitored by the staffs. All animal experiments were performed with approval from the Linfen Central Hospital ethics committee (Permit Number: 2021-29-1).

Conflict of Interest The authors declare no competing interests.

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Greulich S, Mayr A, Gloekler S, Seitz A, Birkmeier S, Schäufele T, et al. Time-Dependent Myocardial Necrosis in Patients With ST-Segment-Elevation Myocardial Infarction Without Angiographic Collateral Flow Visualized by Cardiac Magnetic Resonance Imaging: Results From the Multicenter STEMI-SCAR Project. J Am Heart Assoc. 2019 Jun 18;8(12):e012429.
Han J, Zhang Z, Zhang Z, Yang S. Artemisinin relieves myocardial ischemia-reperfusion injury via modulating miR-29b-3p and hemicentin 1. Front Pharmacol. 2022 Aug 11;13:918966.
GBD 2017 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018 Nov 10;392(10159):1789-1858.
Ermolenko E, Simanenkova A, Voropaeva L, Lavrenova N, Kotyleva M, Minasian S, Chernikova A, et al. Metformin Influence on the Intestinal Microbiota and Organism of Rats with Metabolic Syndrome. Int J Mol Sci. 2022 Jun 20;23(12):6837.
Chen C, Zhang H, Xie R, Wang Y, Ma Y. Gut microbiota aggravate cardiac ischemia-reperfusion injury via regulating the formation of neutrophils extracellular traps. Life Sci. 2022 Aug 15;303:120670.
Bai H, Gu RJ, Chen LY, Qian Y, Yu ML, Xu SL, et al. Electroacupuncture interventions alleviates myocardial ischemia reperfusion injury through regulating gut microbiota in rats. Microvasc Res. 2021 Nov;138:104235.
Lam V, Su J, Koprowski S, Hsu A, Tweddell JS, Rafiee P, et al. Intestinal microbiota determine severity of myocardial infarction in rats. FASEB J. 2012 Apr;26(4):1727-35.
Zhou X, Li J, Guo J, Geng B, Ji W, Zhao Q, et al. Gut-dependent microbial translocation induces inflammation and cardiovascular events after ST-elevation myocardial infarction. Microbiome. 2018 Apr 3;6(1):66.
Inczefi O, Bacsur P, Resál T, Keresztes C, Molnár T. The Influence of Nutrition on Intestinal Permeability and the Microbiome in Health and Disease. Front Nutr. 2022 Apr 25;9:718710.
Legaki E, Gazouli M. Influence of environmental factors in the development of inflammatory bowel diseases. World J Gastrointest Pharmacol Ther. 2016 Feb 6;7(1):112-25.
Yue R, Lv M, Lan M, Zheng Z, Tan X, Zhao X, et al. Irisin protects cardiomyocytes against hypoxia/reoxygenation injury via attenuating AMPK mediated endoplasmic reticulum stress. Sci Rep. 2022 May 6;12(1):7415.
Xiong X, Lu L, Wang Z, Ma J, Shao Y, Liu Y, et al. Irisin attenuates sepsis-induced cardiac dysfunction by attenuating inflammation-induced pyroptosis through a mitochondrial ubiquitin ligase-dependent mechanism. Biomed Pharmacother. 2022 Aug;152:113199.
Zhang W, Chang L, Zhang C, Zhang R, Li Z, Chai B, et al. Central and peripheral irisin differentially regulate blood pressure. Cardiovasc Drugs Ther. 2015 Apr;29(2):121-7.
Fan J, Zhu Q, Wu Z, Ding J, Qin S, Liu H, et al. Protective effects of irisin on hypoxia-reoxygenation injury in hyperglycemia-treated cardiomyocytes: Role of AMPK pathway and mitochondrial protection. J Cell Physiol. 2020 Feb;235(2):1165-1174.
Huangfu LX, Cai XT, Yang JN, Wang HC, Li YX, Dai ZF, et al. Irisin attenuates inflammation in a mouse model of ulcerative colitis by altering the intestinal microbiota. Exp Ther Med. 2021 Dec;22(6):1433.
Metzger CE, Narayanan SA, Elizondo JP, Carter AM, Zawieja DC, Hogan HA, et al.. DSS-induced colitis produces inflammation-induced bone loss while irisin treatment mitigates the inflammatory state in both gut and bone. Sci Rep. 2019 Oct 22;9(1):15144.
Ren YF, Wang MZ, Bi JB, Zhang J, Zhang L, Liu WM, et al. Irisin attenuates intestinal injury, oxidative and endoplasmic reticulum stress in mice with L-arginine-induced acute pancreatitis. World J Gastroenterol. 2019 Dec 7;25(45):6653-6667.
Bi J, Zhang J, Ren Y, Du Z, Li T, Wang T, et al. Irisin reverses intestinal epithelial barrier dysfunction during intestinal injury via binding to the integrin αVβ5 receptor. J Cell Mol Med. 2020 Jan;24(1):996-1009.
Wang H, Zhao YT, Zhang S, Dubielecka PM, Du J, Yano N, et al. Irisin plays a pivotal role to protect the heart against ischemia and reperfusion injury. J Cell Physiol. 2017 Dec;232(12):3775-3785.
Li T, Tan Y, Ouyang S, He J, Liu L. Resveratrol protects against myocardial ischemia-reperfusion injury via attenuating ferroptosis. Gene. 2022 Jan 15;808:145968.
Peng Y, Yan Y, Wan P, Chen D, Ding Y, Ran L, et al. Gut microbiota modulation and anti-inflammatory properties of anthocyanins from the fruits of Lycium ruthenicum Murray in dextran sodium sulfate-induced colitis in mice. Free Radic Biol Med. 2019 May 20;136:96-108.
van Santvoort HC, Besselink MG, Timmerman HM, van Minnen LP, Akkermans LM, Gooszen HG. Probiotics in surgery. Surgery. 2008 Jan;143(1):1-7.
Ji Y, Dai Z, Sun S, Ma X, Yang Y, Tso P, et al. Hydroxyproline Attenuates Dextran Sulfate Sodium-Induced Colitis in Mice: Involvment of the NF-κB Signaling and Oxidative Stress. Mol Nutr Food Res. 2018 Nov;62(21):e1800494.
Chang CJ, Lin CS, Lu CC, Martel J, Ko YF, Ojcius DM, et al. Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota. Nat Commun. 2015 Jun 23;6:7489. doi: 10.1038/ncomms8489. Erratum in: Nat Commun. 2017 Jul 11;8:16130.
Liu Q, Tian H, Kang Y, Tian Y, Li L, Kang X, et al. Probiotics alleviate autoimmune hepatitis in mice through modulation of gut microbiota and intestinal permeability. J Nutr Biochem. 2021 Dec;98:108863.
Xu S, Wu B, Zhong B, Lin L, Ding Y, Jin X, et al. Naringenin alleviates myocardial ischemia/reperfusion injury by regulating the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) /System xc-/ glutathione peroxidase 4 (GPX4) axis to inhibit ferroptosis. Bioengineered. 2021 Dec;12(2):10924-10934.
Zhou W, Cheng Y, Zhu P, Nasser MI, Zhang X, Zhao M. Implication of Gut Microbiota in Cardiovascular Diseases. Oxid Med Cell Longev. 2020 Sep 26;2020:5394096.
Moldovan DC, Ismaiel A, Fagoonee S, Pellicano R, Abenavoli L, Dumitrascu DL. Gut microbiota and cardiovascular diseases axis. Minerva Med. 2022 Feb;113(1):189-199.
Sun L, Jia H, Li J, Yu M, Yang Y, Tian D, et al. Cecal Gut Microbiota and Metabolites Might Contribute to the Severity of Acute Myocardial Ischemia by Impacting the Intestinal Permeability, Oxidative Stress, and Energy Metabolism. Front Microbiol. 2019 Aug 2;10:1745.
Zhou X, Li J, Guo J, Geng B, Ji W, Zhao Q, et al. Gut-dependent microbial translocation induces inflammation and cardiovascular events after ST-elevation myocardial infarction. Microbiome. 2018 Apr 3;6(1):66.
Lee DY, Shin YJ, Kim JK, Jang HM, Joo MK, Kim DH. Alleviation of cognitive impairment by gut microbiota lipopolysaccharide production-suppressing Lactobacillus plantarum and Bifidobacterium longum in mice. Food Funct. 2021 Nov 1;12(21):10750-10763.
Williams NT. Probiotics. Am J Health Syst Pharm. 2010 Mar 15;67(6):449-58.
Chen YT, Chiou SY, Hsu AH, Lin YC, Lin JS. Lactobacillus rhamnosus Strain LRH05 Intervention Ameliorated Body Weight Gain and Adipose Inflammation via Modulating the Gut Microbiota in High-Fat Diet-Induced Obese Mice. Mol Nutr Food Res. 2022 Jan;66(1):e2100348.
Baleeiro FCF, Kleinsteuber S, Sträuber H. Hydrogen as a Co-electron Donor for Chain Elongation With Complex Communities. Front Bioeng Biotechnol. 2021 Mar 31;9:650631.
Singhal R, Donde H, Ghare S, Stocke K, Zhang J, Vadhanam M, et al. Decrease in acetyl-CoA pathway utilizing butyrate-producing bacteria is a key pathogenic feature of alcohol-induced functional gut microbial dysbiosis and development of liver disease in mice. Gut Microbes. 2021 Jan-Dec;13(1):1946367.
Martin-Gallausiaux C, Marinelli L, Blottière HM, Larraufie P, Lapaque N. SCFA: mechanisms and functional importance in the gut. Proc Nutr Soc. 2021 Feb;80(1):37-49.
Arseneault-Bréard J, Rondeau I, Gilbert K, Girard SA, Tompkins TA, Godbout R, et al. Combination of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 reduces post-myocardial infarction depression symptoms and restores intestinal permeability in a rat model. Br J Nutr. 2012 Jun;107(12):1793-9.
Odenwald MA, Turner JR. The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol. 2017 Jan;14(1):9-21.
Qin X, Zou H. The role of lipopolysaccharides in diabetic retinopathy. BMC Ophthalmol. 2022 Feb 22;22(1):86.
Wang L, Yang JW, Lin LT, Huang J, Wang XR, Su XT, et al. Acupuncture Attenuates Inflammation in Microglia of Vascular Dementia Rats by Inhibiting miR-93-Mediated TLR4/MyD88/NF-κB Signaling Pathway. Oxid Med Cell Longev. 2020 Aug 11;2020:8253904.
Liu X, Sun R, Li Z, Xiao R, Lv P, Sun X, et al. Luteolin alleviates non-alcoholic fatty liver disease in rats via restoration of intestinal mucosal barrier damage and microbiota imbalance involving in gut-liver axis. Arch Biochem Biophys. 2021 Oct 30;711:109019.

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Irisin ameliorates myocardial ischemia-reperfusion injury through modulation of gut microbiota and intestinal permeability in rats

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Abstract

Figures

Introduction

Materials And Methods

2.1 I/R rats model establishment and treatment

2.2 Serum Cardiac Troponin I, Creatine phosphokinase, and Lipopolysaccharide measurements

2.3 Histopathological staining

2.4 TUNEL, Immunofluorescence (IF) staining

2.5 Western blot analysis

2.6 Extraction of fecal DNA and high-throughput sequencing

2.7 Bioinformatic analysis

2.8 Statistical Analysis

Result

3.1 Effects of irisin on gut microbial diversity and richness

3.2 Effects of irisin on the gut microbiota composition

3.3 Irisin maintains intestinal integrity in I/R rats

3.4 Effects of irisin on gut inflammation

3.5 Irisin reduces myocardial injury

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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