Cardioprotective Effect of Danhong Injection against Myocardial Infarction in Rats Is Critically Contributed by MicroRNAs

Background Danhong injection (DHI) has been mainly used for the treatment of myocardial infarction, atherosclerosis, and coronary heart disease in clinical practice. Our previous studies have shown that DHI improves ventricular remodeling and preserves cardiac function in rats with myocardial infarction (MI). In this study, we focused on the potential mechanism of DHI in protecting cardiac function in MI rats. Methods Sprague-Dawley rats were subjected to ligation of the left anterior descending coronary artery (LAD) to prepare a myocardial infarction (MI) model. After 14 day DHI intervention, cardiac function was measured by echocardiography and myocardial fibrosis was assessed by Masson staining. Differentiated miRNAs were screened using rat immunopathology miScript miRNA PCR arrays, and their results were verified by RT-PCR, immunofluorescence, and immunoblotting. Results DHI treatment significantly reduced infarct size and improved cardiac function and hemodynamics in MI rats by echocardiography and morphology. miRNA PCR array results showed that DHI reversed 25 miRNAs known to be associated with inflammation and apoptosis. Moreover, the expression of inflammatory factors TNF-α, IL-1β, and IL-6 was significantly reduced in the treated DHI group. Mechanistically, DHI downregulated the inflammatory transcription factor NF-κB (as reflected by inhibition of NF-κB p65 nuclear translocation and phosphorylation of the IκBα). Conclusions DHI is effective in mitigating inflammation associated with MI by preventing NF-κB nuclear translocation and regulating miRNAs, thereby improving cardiac function in myocardial infarction rats.


Introduction
Acute myocardial infarction (AMI) is a common cardiovascular disease with a high mortality rate [1]. Important medical and interventional therapies have been incorporated into the clinical guidelines and shown to reduce the incidence and mortality of acute myocardial infarction in randomized clinical trials [2]. Inflammation is frequently encountered in clinical practice, and various causes can cause myocardial inflammation and lead to defects in the myocardial structure and function [3,4]. e inflammatory response is a prerequisite for scar formation and healing, which can reduce deleterious myocardial remodeling by promoting effective tissue repair after MI [5]. However, overproduction of inflammatory mediators and proinflammatory cytokines can lead to the deterioration of the pathological process. Inhibition of excessive inflammation may allow us to design effective inflammatory-related interventions for treating MI.
MicroRNAs (miRNAs) are endogenous, conserved, single-stranded, small (approximately 22 nucleotides), noncoding RNAs expressed in a cell and tissue-specific manner that regulate gene expression and organ function, including the myocardium [6]. miRNA plays a mediating role in gene expression by combining the 3′-UTR of target genes at the posttranscriptional level by either promoting degradation or inhibiting translation-repress gene expression [7]. Each miRNA can regulate several to hundreds of different target genes by inducing degradation of its target mRNA and inhibiting translation [8,9]. It is widely recognized that miRNAs represent critical regulators of cardiovascular function and show potential as circulating biomarkers of cardiovascular diseases since the discovery of their presence and stability in peripheral blood [10,11], Several studies reported miRNAs have a crucial role in the pathogenesis and progression of heart failure and a huge diagnostic potential in the setting of AMI and its ability to downregulate specific genomic network expressions [12][13][14][15][16]. Increasing evidence shows that miRNAs play functional roles in the development of heart failure and a dysregulation of miRNA expression in the failing or hypertrophic heart [14,[16][17][18][19][20]. Recent research reveals a crucial role of miRNAs as a mediator in the inflammation cascade [21].
More effective and safe anti-inflammatory agents are being investigated, including nonsteroidal anti-inflammatories and traditional Chinese medicine (TCM), with many more compounds under development, such as Xuebijing injection and Shenfu injection have been used for treating inflammatory response, and both of them have resulted in satisfactory effects [22][23][24]. Danhong injection (DHI) is a standard extract from danshen (Salvia miltiorrhiza Bunge, Labiatae) and honghua (Carthamus tinctorius L, Compositae), which has been long used primarily for the treatment of ischemic encephalopathy and cardiac diseases including MI and angina in clinics [25,26]. Our previous study showed that the main components of DHI contain the following substances: danshensu, hydroxysafflor yellow A, 5-hydroxymethyl-2furfural, protocatechuic aldehyde, lithospermic acid, caffeic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, protocatechuic acid, and rosmarinic acid [27]. And research has showed that DHI prevented the lipopolysaccharide-stimulated systemic acute inflammatory reaction via inhibiting the expressions of iNOS, COX-2, IL-1β, IL-6, MCP-1, and TNF-α [28]. is result suggests that DHI may act on several targets to alleviate inflammation.

Quality Control of DHI.
According to the National Drugs surveillance administrative bureau standard WS-11220, the total amount of danshensu (molecular formula: C 9 H 10 O 5 ) and protocatechuic aldehyde (molecular formula: C 7 H 6 O 3 ) should not be lower than 0.5 mg in 1 mL DHI analyzed by high-performance liquid chromatography (HPLC) as a reference in 1 mL injection. Simultaneously, the content of the total flavonoids determined by visible light spectrophotometry should not be lower than 5.0 mg/mL against rutin (molecular formula: C 27 H 30 O 16 ) as a reference in 1 ml injection [30].
As early as 2012, our laboratory has established a dualstandard quality assessment of DHI [27]. HPLC fingerprinting of DHI has been performed on the ACQUITY UHPLC BEH C18 column (2.1 × 150 mm 2 , 1.7 m) and an C18 guard column. e quality control of the DHI method is as follows. e flow rate was 0.3 mL/min. e column temperature was 30°C. e mobile phase comprised (A) aqueous phosphoric acid (0.2%, v/v) and (B) acetonitrile using a gradient elution of 5-5% B at 0-3 min, 20-20% B at 15-20 min, 27-27% B at 25-28 min, and 27-70% B at 28-30 min, and the reequilibration time of gradient elution was 2 min. Moreover, the detection wavelength was set at 280 nm [31].

Animals.
Male Sprague-Dawley rats, weighing 220-250 g, was purchased from Beijing HFK Bioscience. Co., Ltd. e rats were raised at 12 h dark/light cycle with a temperature of 22 ± 2°C and humidity of 40 ± 5%. Standard rat chow and clean drinking water were provided ad libitum.
e study was carried out in accordance with the recommendations in the guidance for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology, China. All animal experiments and procedures were approved by Tianjin University of Traditional Chinese Medicine (TCM) Laboratory Animal Ethical Committee (TCM-LAEC2017026), and all procedures were performed to reduce the number and animals and suffering during the experiments.
Rats were anesthetized with chloral hydrate (5%, peritoneal injection, 300 mg/kg). Adequacy of anesthesia was controlled by monitoring corneal reflex and the lack of response to toe-pinching. Euthanasia was performed by excessive inhalation of isoflurane. Death was monitored by the cardiac activity and respiration.

Inducing Myocardial Infarction in a Rat and Drug
Administration. For the MI rat model, the left anterior descending (LAD) coronary artery was subjected to ligation according to the previous studies [32], and the specific process was carried out as previously reported [29].

Echocardiographic and Hemodynamic Assessment of Left Ventricular
Function. e functionality of the left ventricle was evaluated at 14 days after MI, using a Vevo 2100 Ultrahigh resolution small animal ultrasound imaging system in real time (VisualSonicsVevo 2100, Canada), as previously described [33]. e cannulation was done to the left ventricle through the right carotid artery, which was connected to a biofunction experiment system MP100-CE (BIOPAC systems Inc., Santa Barbara, California, USA) at the end of the echocardiography experiment. e detailed procedure is described in our earlier report [29].

MicroRNA PCR Array.
e total RNA was extracted from the LV samples (Infarct border zone, 30 mg/each rat) with miRNeasy Mini kit (Qiagen, Germany) according to the manufacturer's instructions. e RNA samples (2 μg) were reverse transcribed to cDNA with the miScript II RT kit (Qiagen, German). e reaction system and conditions are shown in Table 1.
Using miScript PCR Starter Mix Reagent kits (Qiagen) and miScript miRNA PCR arrays (rat immunopathology miRNA PCR array: MIRN-104Z) to run the Q-PCRs, miRNAs expression was confirmed by specific primers. e reaction system and cycling conditions are shown in Tables 2 and 3.

Quantitative Real-Time PCR.
Real-time PCR in a Ste-pOne Real-Time PCR system (Applied Biosystems) was used to determine the cardiac mRNA expression of IL-1β, TNF-α, caspase-3, Bcl-2, and GAPDH. e sequences of the sense and antisense primers used for amplification are shown in Table 4.

Serum Biochemical Indices.
At the 7th and 14th day after surgery, tagged as the inflammatory reaction period, serum was also collected from the rats. e levels of inflammatory factors which involve TNF-α, IL-1β, and IL-6 were measured by commercially available ELISA kits according to the manufacturer's instructions.

Western Blot Analysis.
Myocardial tissues were cut from the infarct border zone. Cytosolic and nuclear proteins were extracted from the infarct border zone of the heart tissue using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer's instructions ( ermo Scientific, USA). Proteins were separated by SDS-PAGE (8-10%) and probed with different primary antibodies against p-P65, NF-κB, p-IκB-α, GAPDH, and Lamin B.

Histopathological Examination.
Myocyte size and interstitial fibrosis were shown by pathological section.

Evidence-Based Complementary and Alternative Medicine
Pathological examination was performed using the paraffin sections and stained with hematoxylin and eosin (H&E) and Masson trichrome. Infarct size measurements were performed by measuring the midline length [34].

Statistical Analysis.
All data in the experiment were expressed as mean ± SD. Data analyses were performed using the SPSS 17.0 software package. Statistical analysis was assessed by the analysis of variance (ANOVA) followed by the LSD test or Dunnett's t-test for multiple comparisons. e differences were considered statistically significant at the value of p < 0.05.

Treatment with DHI Improves Cardiac Performance and
Hemodynamics in the MI Rat Model. Cardiac function was measured 14 days afterward via echocardiographic assessments (LVEF, LVFS, LVAWs, LVIDs, LVVOLs, and Tei value). LVEF, LVFS, and LVAWs were significantly greater in rats administered with DHI and valsartan than in those treated with saline, while LVIDs, LVVOLs, and Tei value were significantly smaller (Figures 1(a)-1(f )). e representative echocardiograms in different groups are presented in Figure 1(m). Hemodynamic parameters measured via an intracardiac Millar catheter are presented in Figures 1(g)-1(j). Rats with MI developed systolic dysfunction, as evidenced by significantly decreased LVSP and the maximum rate of rising in LV pressure (+dp/dt max), also exhibited a severe diastolic dysfunction, as defined by elevated LVEDP and the maximum rate of decline in LV pressure (− dp/dt max) in the model group than the sham group. DHI and valsartan administration significantly improved systolic cardiac function, enhancing LVSP and +dp/dt max, and provided beneficial effects on the diastolic function by reducing LVEDP and − dp/dt max compared to saline-treated rats (p < 0.05). DHI significantly ameliorates cardiac function, by increasing SW (5951.33 ± 1313.81 compared to the model 3307.83 ± 1304.61 mmHg * μL; p < 0.01), whereas HR did not differ significantly between all groups at 2 weeks after MI (Figures 1(k) and 1(l)).

Myocardium Histology.
We directly observed the effect of DHI on the myocardial structure and gross morphology after myocardial infarction by H&E and Masson staining ( Figure 2). Morphological observation of H&E staining showed increase in myocardial hypertrophy and cell gap, loose of the structural arrangement of myocardial cells, rupture of myocardial fibers, and inflammatory cell infiltration, while a relative slighter condition in drug-treated groups was observed (Figure 2(a)). Less myocardial fibrosis is found in the heart after DHI and valsartan treatment as demonstrated by Masson staining (Figure 2(b)). Interstitial collagen density was markedly increased in the LV myocardium of the rat model than the sham group. Treatment with DHI significantly reversed this effect. e statistical infarction ratio was conducted by the application of the midline method. Compared with the model group, ratio of infarct was markedly decreased in the DHI and valsartan treatment group (Figure 2(c)).

MircoRNA Expression Pattern in DHI-Treated MI Rats
Model.
e rat immunopathology miScript miRNA PCR array was applied to screen for differential miRNA, and the miRNA PCR array result revealed that there were 5 upregulated and 15 downregulated miRNAs genes (fold change > 2) in the model group compared to the sham group and 25 upregulated miRNAs genes (fold change > 2) in the DHI-treated group compared to the model group. ese significant differences in miRNAs are related to the immune response and inflammation, as shown in Tables 5 and 6.

Role of miRNAs in the MI Rat Model as Revealed in
Microarray-Based Bioinformatics Prediction. In order to obtain a better overview of miRNA expression signature, we further performed scatter diagram and unsupervised hierarchical clustering heat map analysis of differentially expressed miRNA by normalized probe signal values (Figures 3(a) and 3(b)). First and foremost, the miRNA PCR array was used to analyze miRNA expression profiles in the model and DHI group. Fold change > 2 and p value < 0.05 between model and DHI group were set as the criteria for filtering differently expressed miRNAs. e scatter diagram result revealed that the expression of 25 miRNAs was strongly upregulated under 14-day DHI treatment. e heat map showed differentially expressed miRNA profiling.
According to the selected differentially expressed miRNA (fold change > 2), we have carried out the target gene prediction through the GeneSpring and microRNAorg database.
e p value of the predicted target gene was calculated by hypergeometric distribution. GO and KEGG analysis was performed on the target gene with p ≤ 0.05 using David software, and a miRNA-mRNA network was constructed.
In order to predict which possible functions and biological pathways were affected in the MI rat model and DHI group, we performed gene ontology (GO) and KEGG pathways analysis of 19 miRNAs which significantly changed after pretreatment with DHI. Bioinformatics analysis predicted that 19 miRNAs up-or downregulated 54 GOs significantly (refer miRNA-GO-network (Figure 3(c)) for more detailed information). Moreover, miRNA-KEGG signal pathway analysis revealed that the molecular pathways regulated by the differential miRNAs in the expression level mainly included the calcium signaling pathway, apoptosis, glycosylphosphatidylinositolanchor biosynthesis, valine, leucine, and isoleucine degradation, PPAR signaling pathway, VEGF signaling pathway, natural killer cell-mediated cytotoxicity, purine metabolism, butanoate metabolism, amyotrophic lateral sclerosis, fatty acid metabolism, and long-term potentiation (Figure 3(d)). e miRNA-mRNA network was constructed based on the relationship between differentially expressed miRNAs and their target genes. e results show that the differential miRNAs such as let-7a-5p, miR-103-3p, miR-142-5p, miR-143-3p, miR-185-5p, miR-186-5p, miR-26b-5p, miR-205, and miR-207 constituted intensive molecular networks and participated in the regulation of the immune response, e.g., TNF-α, IL-1β, and caspase-3 (Figure 3(e)), Table 6). is result poses an important question which led to the further experiment.

Effect of DHI on Prediction of Target Gene Expression.
Based on miRNA PCR array results, hearts taken from rats in each of the experimental group were used for the confirmation of TNF-α, IL-1β, caspase-3, and Bcl-2 on the mRNA level (Figure 4). e RT-PCR result showed that mRNA expressions of TNF-α, IL-1β, and caspase-3 were

Effects of DHI on the Serum Level of Inflammatory
Cytokines. To further validate the prediction results, TNF-α, IL-1β, and IL-6 were measured in the serum at 14 days postsurgery. A shown in the results displayed in Figure 5, rats with MI had a higher serum level of TNF-α, IL-1β, and IL-6 concentration than that in the sham group (p < 0.05). ese cytokines levels in the DHI group were significantly decreased (p < 0.05 and p < 0.01), while the valsartan group showed the same effect, but there was no significant difference in IL-1β.

DHI Modulates NF-κB Signaling Pathway in the MI Rat
Model.
e effect of DHI on NF-κB pathway activation was examined in the cytoplasm and nucleus of cardiomyocytes from MI rats. e cytosolic phosphorylation of NF-κB and IκB-α was significantly enhanced in the model group; however, this change was notably inhibited by DHI and valsartan treatment (Figure 6(a)). Additionally, the nuclear expression of NF-κB was observed. Interestingly, MI not only increased the phosphorylation of NF-κB in the cytoplasm but also markedly enhanced the expression of NF-κB in the nuclear (p < 0.01, the model group compared with the sham group). However, Figure 6(b) showed that DHI and valsartan obviously inhibited the MI induced changes (p < 0.01, compared with the model group).

Discussion
A large number of evidence suggest that miRNAs play a critical role in diverse biological processes, such as embryogenesis, differentiation, carcinogenesis, immune system,  Evidence-Based Complementary and Alternative Medicine inflammation, and viral infection, as well as involved in the physiological and pathophysiological processes such as cardiomyocytes apoptosis, angiogenesis, myocardial hypertrophy, ventricular remodeling, and heart failure in the cardiovascular system [6,[35][36][37]. Robust evidence has shown that regulation of miRNAs has an inseparable relationship with inflammation. More and more miRNA have been reported to be involved in the regulation of the immune system, such as the release of inflammatory mediators [35]. Firstly, we observed that DHI could obviously improve the LVEF and LVFS and increase the LVSP and ±dp/dt max in MI rats, which show that DHI can improve cardiac function and hemodynamics in rats after MI. In order to explore the mechanism of DHI improving cardiac function in MI rats, we used microRNA PCR array to predict and analyze its target.
is result is consistent with the previous reports that miRNAs can also act directly on the mRNA of cytokines to attenuate the inflammatory response and maintain the body immune balance [38].
Another evidence is that TNF-α binds to homologous receptors to raise and activate caspase-8 in animal models  and in humans subjects, which in turn activates the main executioner of apoptosis and caspase-3 [39][40][41]. Moreover, miRNA-KEGG signal pathway analysis revealed that the differential miRNAs in the expression level regulating the molecular pathways mainly included the calcium signaling pathway, apoptosis, glycosylphosphatidylinositol-anchor biosynthesis, valine, leucine, and isoleucine degradation, PPAR signaling pathway, VEGF signaling pathway, natural killer cell-mediated cytotoxicity, purine metabolism, butanoate metabolism, amyotrophic lateral sclerosis, fatty acid metabolism, and long-term potentiation. DHI regulates these signaling pathways such as VEGF and apoptosis signaling pathway and has also been confirmed (data not shown). erefore, our results suggest that DHI could protect the heart function of rats with MI by regulating miRNAs. is is a new mechanism by which DHI improves cardiac function in rats with myocardial infarction.
Another important evidence showed that IL-6 is the target for the Let-7 family of miRNAs, while Let-7 itself is also negatively regulated by TLR/NF-κB [42,43]. In the present study, DHI can significantly inhibit NF-κB signaling pathway. It is well known that the transcription factor NF-κB is closely involved in the inflammatory cascade, manifested by increased NF-κB nuclear translocation, and promotes phosphorylation or degradation of IκB-α. NF-κB is a central transcriptional effector of inflammatory signaling which is involved in the regulation of cellular proliferation, differentiation, and apoptosis [31,44]. Five subunits (RelA [p65], RelB, c-Rel, NF-κB 1 [p50], and NF-κB 2 [p52]) comprise the NF-κB family [45]. It has been reported that p65 and p50 heterodimers are the main types of NF-κB in cells and also the two most intensively studied subtypes of the NF-κB family at the moment [46,47]. NF-κB activation and its subsequent nuclear translocation after MI trigger transcription of a large portfolio of genes including inflammatory cytokines (TNF-α, IL-1β, and IL-6), CXC, CC chemokines, and adhesion molecules [44]. Interestingly, our result agrees with the preservation of cardiomyocytes by DHI against inflammatory injury through the inhibition of the nuclear transcription of NF-κB and IκB-α phosphorylation. is indicates that DHI may have a pivotal role in alleviating the inflammatory injury caused by MI. ese results provide evidence that the anti-inflammatory activity of DHI could be due to the regulation of the inflammation mediators via NF-κB pathway at the MI process.
Although this study elucidates that DHI could resist myocardial infarction to some extent by regulating micro-RNAs, there are still many miRNAs that need further research. In particular, how DHI regulates miRNAs to improve cardiac function is a problem worthy of further study.

Conclusion
In summary, our study confirmed that DHI ameliorates cardiac function and reduces myocardial infarct size via its anti-inflammatory effect through inhibiting NF-κB pathway and regulating miRNA. In brief, DHI prevents the deterioration of inflammatory response by inhibiting nuclear translocation of NF-κB and modulating inflammation-associated miRNA (such as miR-103-3p, miR-130a-3p miR-331-3p, miR-205, and miR-26b-5p), as well as regulating some target genes (TNF-α, IL-1β, and caspase-3) of the miRNA in the infarcted marginal zone. DHI inhibits myocardial cell apoptosis and promotes angiogenesis, thereby reducing infarct size and improving cardiac function. ese findings may contribute to the understanding of molecular mechanisms involved in cardioprotection of DHI and provide novel insights into a future therapeutic strategy for early myocardial ischemia event.
Data Availability e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
JC and JW designed the animal model, performed echocardiographic assessment of left ventricular function, western blot, and real-time PCR, and wrote the manuscript. JO analyzed the results of the miRNA experiment. ZY, HZ, and JY provided drugs and discussed interpretation. JN performed the histopathological examination. LL performed the hemodynamic assessment of left ventricular function. XG and GF conceived and designed the animal experiment. All the authors read and approved the final manuscript.