Curcumin Ameliorates Cardiac Fibrosis by Regulating Macrophage-Fibroblast Crosstalk via IL18-P-SMAD2/3 Signaling Pathway Inhibition

Ethnopharmacological relevance: Curcumin is a bright yellow chemical produced by plants of the Curcuma longa species. Chemically, curcumin is a diarylheptanoid, belonging to the group of curcuminoids. The therapeutic potential of curcumin has been widely investigated, including its utilization in various of cardiovascular diseases. However, its effect in cardiac remodeling post myocardial infarction and underlying mechanism remains to be uncover. Aim: To evaluate the therapeutic effect and underlying mechanism of curcumin on cardiac fibrosis after myocardial infarction via macrophage-fibroblast crosstalk. Methods: Male C57BL/6 (C57) mice were subjected to left anterior descending coronary artery ligation to establish myocardial infarction and intragastrically fed vehicle or curcumin (50 mg/kg or 100 mg/kg) for 4 weeks. In parallel, neonatal rat cardiac fibroblasts were isolated and co-cultured with liposaccharide (LPS− or LPS+) curcumin-treated macrophages, followed by TGF-β stimulation for 24 h. Cardiac function was determined by 2-dimensional echocardiography, and cardiac fibrosis was measured by picrosirius red staining. Apoptosis of macrophages was investigated by flow cytometry; all pro-fibrotic protein expression (EDA-Fibronectin, Periostin, Vimentin, and α-SMA) as well as TGF-βR1 downstream signaling activation reflected by phosphorylated SMAD2/3 (p-SMAD2 and p-SMAD3) were demonstrated by western blotting. Results: Curcumin significantly ameliorated the inflammation process subsequent to myocardial infarction, reflected by decreased expression of CD68+ and CD3+ cells, accompanied by dramatically improved cardiac function compared with the placebo group. In addition, cardiac fibrosis is inhibited by curcumin administration. Interestingly, no significant reduction in fibrotic gene expression was observed when isolated cardiac fibroblasts were directly treated with curcumin in vitro; however, pro-fibrotic protein expression was significantly attenuated in CF, which was co-cultured with LPS-stimulated macrophages under curcumin treatment compared with the placebo group. Mechanistically, we discovered that curcumin significantly downregulated pro-inflammatory cytokines in macrophages, which in turn inhibited IL18 expression in co-cultured cardiac fibroblasts using bulk RNA sequencing, and the TGF-β1-p-SMAD2/3 signaling network was also discovered as the eventual target downstream of IL18 in curcumin-mediated anti-fibrosis signaling. Conclusion: Curcumin improves cardiac function and reduces cardiac fibrosis after myocardial infarction. This effect is mediated by the inhibition of macrophage-fibroblast crosstalk in the acute phase post-MI and retrained activation of IL18-TGFβ1-p-SMAD2/3 signaling in cardiac fibroblasts.

Results: Curcumin significantly ameliorated the inflammation process subsequent to myocardial infarction, reflected by decreased expression of CD68 + and CD3 + cells, accompanied by dramatically improved cardiac function compared with the placebo group. In addition, cardiac fibrosis is inhibited by curcumin administration. Interestingly, no significant reduction in fibrotic gene expression was observed when isolated cardiac fibroblasts were directly treated with curcumin in vitro; however, pro-fibrotic protein expression was significantly attenuated in CF, which was co-cultured with LPSstimulated macrophages under curcumin treatment compared with the placebo group.

INTRODUCTION
Cardiovascular diseases, especially ischemic heart disease, remain the leading cause of death worldwide. Although early reperfusion therapy for acute myocardial infarction (MI) can effectively salvage ischemic myocardium, a considerable portion of cardiomyocytes may still experience irreversible necrosis and loss, followed by ventricular remodeling and cardiac insufficiency, which compromises the long-term survival of patients with MI (Tallquist and Molkentin, 2017). Hence, it is crucial to develop effective therapies for adverse cardiac fibrosis subsequent to MI.
Monocytes and macrophages play a critical role in regulating fibrotic responses in various tissues, including cardiac tissue after ischemic stimulation (Wynn and Vannella, 2016). The normal adult mammalian myocardium contains a relatively small population of resident macrophages (Epelman et al., 2014) , (Heidt et al., 2014) which have been suggested to play a role in cardiac homeostasis by facilitating atrioventricular conduction (Hulsmans et al., 2017). Following injury, resident cardiac macrophages derived from embryonic yolk sac cells are replaced by an abundant population of monocyte-derived macrophages (Epelman et al., 2014), recruited through the activation of chemokine-dependent pathways (Dewald et al., 2003). Macrophages in injured hearts are highly heterogeneous and exhibit functional and phenotypic versatility that enables them to participate in a wide range of processes, including inflammation regulation, fibrosis, matrix remodeling, angiogenesis, and regeneration (Honold and Nahrendorf, 2018). Thus, subsets of activated macrophages may regulate fibrosis by serving as a major source of cytokines and growth factors with fibrogenic properties, secretion of proteases that participate in matrix remodeling, and production of matricellular proteins. Proinflammatory cytokines (such as IL-1β, IL-6, and TNF-α) secreted in many cardiac fibrotic conditions may promote a fibrogenic macrophage phenotype by inducing transcription of members of the TGF-β superfamily.
Turmeric is acquired from Curcuma long L, a tuberous herbaceous perennial plant with yellow flowers and wide leaves, which is a member of ginger family and grows in tropical climate (Prasad et al., 2014). Unlike cinnamon, turmeric has not any different kinds. Curcumin is a symmetric molecule consisting of two similar aromatic rings that contain O-methoxy phenolic groups connected by a carbon linker with an α, β-unsaturated β-diketone moiety (Priyadarsini, 2014). Curcumin's health benefits has been well-established, including anti-tumor, anti-viral, antioxidative stress, anti-inflammatory, anti-microbial, hypoglycemic etc. Therapeutically, curcumin exhibits promising potential in preclinical as well as clinical studies and is currently in human trials for a variety of conditions, including metabolic syndrome, nonalcoholic fatty liver disease, atherosclerosis, liver cirrhosis, depression, psoriasis, and Alzheimer's disease (Kocaadam and Şanlier, 2017). The immunomodulatory functions of curcumin arise due to its interactions with cellular and molecular components during inflammatory reactions. Dietary exposure to 40 mg/kg curcumin for 5 weeks showed enhanced IgG levels in rats, suggesting an improvement in immune function after curcumin intervention (South et al., 1997). Curcumin has also been shown to regulate macrophage polarization by increasing the M2 phenotype marker CD163 together with the anti-inflammatory cytokine IL-10 and decreasing the M1 phenotype marker CD86 along with the pro-inflammatory cytokines TNF-α and IL-6 (Li et al., 2017).
Inflammasomes play an important role in mediating fibrosis in cardiac fibroblasts, which are commonly initiated through NLRP3 activation and response and in turn are amplified by IL-18 secretion (Elliott and Sutterwala, 2015). Due to the increased IL-18 level, the synthesis and secretion of TGF-β1 and cytokines can be promoted through autocrine signaling, leading to further inflammatory activation and phosphorylation of SMAD2/3, which initiates fibrogenesis waterfall reactions.
To summarize, our study aimed to unveil the therapeutic effect of curcumin in alleviating IL-18-p-SMAD2/3-induced cardiac fibrosis subsequent to MI, which is mediated by the inhibition of inflammation-induced macrophage-fibroblast crosstalk.

Animals and Experimental Design
Forty male C57/BL/6J mice weighing 20-25 g were purchased from the Zhejiang Experimental Animal Center (Hangzhou, China). All experiments were approved by the Ethical Committee of Zhejiang University and all surgical procedures were performed by experienced technician sin a blinded manner. Mice were acclimatized to the standard conditions with 12 h lighting cycle, 25 ± 2°C temperature, free access to water and standard chow for 1 week. Then, they were sorted into four groups of 10 mice per group. Group 1 (sham) received dimethylsulfoxide (DMSO-saline) for 28 days intragastrically (i.g.) as a vehicle for curcumin. Group 2 (MI + DMOS) was treated with MI.Group 3 was treated with curcumin 50 mg/kg/day i.g. for 28 days after MI. Group 4 was treated with curcumin 100 mg/kg/day i.g. for 28 days after MI. The appropriate dose (50 mg/kg and 100 mg/kg) was selected (Wang et al., 2019;He et al., 2020). MI was induced by ligation of the left anterior descending coronary artery. The MI model was established as described previously (Hu et al., 2008).
Two-dimensional B-mode and M-mode measurements in the long-axis view level include left ventricular end-diastolic dimension (LVID,d), left ventricular end-systolic dimension (LVID,s), interventricular septal wall thickness in diastole (IVS,d) and in systole (IVS,s), and left ventricular posterior wall thickness in diastole (LVPW,d) and in systole (LVPW,s). Left ventricular ejection fraction and fractional shortening were automatically calculated by the echocardiographic system (Xiao et al., 2018).

Immunohistochemical Staining
Mouse hearts 7 days post-MI were dehydrated in 30% sucrose solution, embedded in Tissue-Tek OCT compound, snap-frozen in dry ice, and then cut into 7 μm sections. The sections were then stained with a In Situ Cell Death Detection Kit (Roche Applied Science, CH), CD3, CD68, Troponin I (Abcam, United Kingdom), and DAPI (Vector Laboratories, Burlingame, CA, United States).
Mouse hearts 28 days post-MI were fixed in 10% formalin-PBS, then paraffin embedded, and cut into 3 μm sections. After deparaffinization, rehydration and tissue antigen recovery, the sections were stained with Periostin (R&D, United States), Troponin I (Abcam, United Kingdom), and DAPI (Vector Laboratories, Burlingame, CA, United States).

Picro Sirius Red Staining
After deparaffinization, sections were stained with Sirius Red. Tissue damage was scored by calculating the scar circumference, including both internal and external scar diameters. The sirius red-stained sections were scanned with a microscope digital camera (Olympus Instrument, United States), and Biotechnologies, China). The percentage of fibrotic area was calculated as the mean value of the endocardial and epicardial length of the whole fibrotic area in proportion to the mean length of the endocardial and epicardial left ventricle using Image using Image Pro Plus software version 6.0.

Macrophage Culture and Drug Treatment
Mouse macrophage-like Raw 264.7 cells were acquired from the ATCC and cultured in DMEM with 10% FBS and 1% Penicillin/ Streptomycin at 37°C with 5% CO 2 . After 2 days, the medium was replaced, and nonadherent cells were discarded.

Cytotoxicity Test
Raw 264.7 cell viability was examined using the CCK-8 assay (Bio-sharp, China) in accordance with the manufacturer's protocols. Cells were treated with liposaccharides (LPS 1 μg/ ml) or LPS with curcumin at different concentrations (0.1, 1, 10, 20, and 50 μM) and the appropriate dose (10μM, 20 μM) was selected refer the previous studies (Gao et al., 2015;Chun-Bin et al., 2020). Then Cells were seeded in a 96-well plate at a density of 8×10 3 cells/well. Following treatment, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. Survival Ratio was calculated according to the following equation: cell survival [(As-Ab)/(Ac-Ab)] ×100%, where As treated group, Ac normal group, and Ab vehicle control group. The absorbance of each well was measured at 450 and 630 nm using a microplate reader (Spark TECAN, Switzerland). All data were calculated from triplicate samples.

Cell Apoptosis
Raw 264.7 cells were treated with 1 μg/ml LPS or LPS with curcumin (10 and 20 μM). Cell apoptosis was evaluated by flow cytometry with Annexin V-FITC and PI staining (CytoFlex, Beckman Coulter, Germany). The flow cytometry assay was examined in accordance with the manufacturer's protocols.

Cell Co-culture Scheme
NRCFs were isolated from P0-P3 neonatal rats using the Neonatal Heart Dissociation Kit mouse and rat (Miltenyi, United States) in accordance with the manufacturer's protocols. Raw 264.7 cells were first seeded in 0.4 μm Transwell chamber (#3412), after treatment in low-glucose DMEM with serum deprivation overnight, NRCFs were stimulated with 10 ng/ml TGF-β and then co-cultured with 1 μg/ml LPS or 1 μg/ml LPS with 20 μM curcumin, respectively.

Plasmid Transfection
NRCFs in each group were seeded in a 6-well plate at 2 × 10 5 cells per well and cultured in FBS free medium overnight. NRCFs were transfected for 48 h with 2 μg of the IL18 mimic (IL-18 rat, Shanghai GenePharma Co., Ltd, China) to overexpress IL18 using X-tremeGENE HP DNA Transfection Reagent according to the manufacturer's instructions. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to quantify IL-18 transfection efficiency. Conditioned medium was collected from transfected cell medium and analyzed according to the manufacturer's instructions.

Statistical Analysis
All results were expressed as value ±standard error of the mean (SEM). Significant differences between two groups were determined by the Student's t-test, and One-way ANOVA test was used for multiple comparisons, two-way ANOV test was conducted in multiple group with different time points. p < 0.05, p < 0.01, was considered statistically significant present *, **, respectively. Statistical calculations were carried out using GraphPad Prism 9.0. The sample size was ≥5 in each group for in vivo animal studies, and ≥3 in each group for in vitro studies.

Curcumin Resists Inflammation Response Post-MI and Supports Subsequent Cardiac Function
To test the effect of curcumin on MI, we performed MI surgery by permanent ligation of the left anterior descending artery followed . Results are mean with SEM, NS no significance between groups, *p < 0.05, **p < 0.01, n.
Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 784041 6 by intragastric administration of curcumin for 7 days or 28 days. In parallel, a placebo post-MI group was also established. At 7 days post-MI surgery, we discovered significantly inhibited inflammation activation reflected by reduced CD68 + and CD3 + cells detected in the peri-infarcted area in the curcumin group compared with the placebo group, while no difference in CD68 + and CD3 + cell counts was observed between the high-dose curcumin group (100 mg/kg) and the low-dose curcumin group (50 mg/kg) (Figures 1A-D). In addition, cardiac function in the MI + curcumin group exhibited significant improvement compared with the placebo group 1 month after MI, with no obvious change observed 7 days post-MI between the two groups, as demonstrated by ejection fraction (EF%) and fraction shortening (FS%) (Figures 1E-G), in addition, the chamber of left ventricle in systolic phase was dramatically decreased in MI + curcumin group compared with MI group (LVID s), while no significant change were detected between groups in diastolic phase (LVID d) (Figures 1H,I). To summarize, the administration of curcumin after MI significantly ameliorated inflammation in the acute phase; however, curcumin exerted a protective effect by preserving long-term cardiac function only after MI, which suggested that the reduced inflammation activation might be related to adverse cardiac remodeling mediated by curcumin intake.

Curcumin Ameliorates Cardiac Fibrosis and Reverses Adverse Remodeling Post-MI
As previously indicated, curcumin delivery significantly improved long-term cardiac function after MI in C57 mice.
To determine what yields this beneficial effect, we conducted TUNEL and picro sirius red staining and, surprisingly, discovered no significant reduction in anti-apoptotic effect on cardiomyocyte within border area in the curcumin-treated group compared with the placebo group 7 days after MI (Supplementary Figure S1A,B). However, we observed dramatically reduced scar formation reflected by picro sirius red staining in both scar circumference and infarct size dimension in curcumin group compared with the placebo group (Figures 2A-C). More importantly, we discovered significant ameliorated fibrosis within noninfarcted area in MI + curcumin group compared with MI group using the dosage of 100 mg/kg ( Figures 2D,E), indicating the robust anti-fibrotic role of curcumin post MI. In addition, we conducted immunostaining on MI segments using periostin to verify cardiac myofibroblast enrichment and collagen deposition, revealing significantly decreased periostin + cell expression within MI segment in Curcumin group ( Figures 2F,G), western blot results also confirmed the therapeutic utilization of Curcumin in inhibiting pro-fibrotic protein expression as displayed by ameliorated Periostin, Vimentin, and α-SMA expression ( Figure 2H-K). The results presented above indicate that the use of curcumin following MI significantly improved cardiac function by inhibiting excessive collagen deposition and scar formation.

Curcumin Promotes Macrophage Apoptosis Under LPS Stimulation in vitro Accompanied by Inhibited Pro-inflammatory Cytokine Secretion
It is known that LPS significantly activate inflammation and mobilize macrophage proliferation as well as M1 polarization. To further investigate the mechanism of curcumin in antiinflammatory processes, we performed LPS stimulation on macrophage in vitro for 24 h followed by Curcumin administration, CCK8 test revealed dramatic inhibited macrophage proliferation when dosage was 20 uM, indicating Curcumin might exerted anti-proliferation or pro-apoptotic effect in macrophages under LPS stimulation ( Figure 3A). Flow cytometry was conducted to determine whether curcumin promoted macrophage apoptosis under LPS stimulation. As shown in Figures 3B,C, curcumin significantly increased macrophage apoptosis under LPS stimulation compared with LPS only also using 20 uM dosage. In addition, the cleaved-caspase three protein level, which acts as a marker of apoptosis, was also significantly up-regulated in the curcumintreated macrophages with LPS group compared with LPS only reflected by western blot (Figures 3D,E). Furthermore, macrophage activation could induce the secretion of proinflammatory cytokines such as IL-6, IL1β, and TNF-α to initiate an immune response to ischemic injury, thus aggravating cardiomyocyte apoptosis; however, curcumin treatment significantly inhibited pro-inflammatory cytokine secretion in macrophages with LPS group compared with LPS only, as reflected by reduced IL-6, IL-1β, and TNF-α secretion especially at 20 uM concentration ( Figures 3F-H). These data indicate that the administration of curcumin on macrophages under LPS stimulation exerted both pro-apoptotic effects in macrophages and anti-inflammatory effects by reducing cytokine release from macrophages, which could be greatly beneficial post-MI.

Curcumin Exerts Anti-Fibrotic Effects via Macrophage-Fibroblast Crosstalk
As previously mentioned, curcumin has also been shown to regulate macrophage polarization by increasing antiinflammatory cytokine IL-10 levels and decreasing the M1 phenotype marker CD86 along with the pro-inflammatory cytokines TNF-α and IL-6. To validate whether curcumin exerted an inhibitory effect on cardiac fibrosis by alleviating inflammation-induced fibrosis or by directly suppressing cardiac fibroblast trans-differentiation and collagen secretion, we co-cultured macrophages under LPS stimulation with isolated primary neonatal rat cardiac fibroblasts in vitro followed by TGF-β stimulation in NRCF for 24 h, Surprisingly, we discovered that curcumin administration in macrophages with LPS stimulation significantly mitigated collagen synthesis from co-cultured NRCF, which was revealed by pro-fibrotic protein expression (α-SMA, Vimentin, Periostin, and EDA-Fibronectin) ( Figure 4A,C-F). In contrast, we did not observe a significant change in fibrosis protein expression when curcumin Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 784041  was directly added to NRCF treated with TGF-β for 24 h compared with the TGF-β only group ( Figure 4B,G-J).
Mechanistically, we discovered decreased phosphorylation of SMAD2/3 in the curcumin-macrophage co-cultured NRCF group compared to NRCF without curcumin treatments, as reflected by the p-SMAD2/3 to total SMAD2/3 ratio ( Figure 4K-M), importantly, we also discovered downregulated phosphorylation of SMAD2/3 in hearts from post-MI with curcumin treated group compared with MI group, indicating the consistent role in anti-phosphorylation of curcumin towards SMAD2/3( Figure 4N-P). In summary, the administration of curcumin only inhibited LPS-stimulated macrophage-fibroblast crosstalk induced excessive collagen deposition, this effect is mediated by the inhibition of SMAD2/3 phosphorylation, while in NRCF with TGF-β stimulation, curcumin delivery was unable to reverse established pro-fibrotic protein expression.

Curcumin Alleviates Cardiac Fibroblast Trans-differentiation by Inhibiting IL-18 Expression Promoted by Macrophage-Fibroblast Crosstalk
To elucidate the detailed mechanism of the identified curcumin-mediated anti-fibrosis effect, we performed bulk mRNA sequencing in NRCF co-cultured with curcumintreated macrophages and NRCF co-cultured with PBStreated macrophages; 776 genes were downregulated and 1,467 genes were upregulated in the curcumin group compared with the PBS group ( Figures 5A,B). Gene enrichment from GO pathway analysis revealed that both immune and defense response signaling were mostly activated and altered between the two groups and that these two signaling pathways were classified and regulated by defense response regulation, indicating the pivotal utilization of curcumin in alleviating an overreactive immune response ( Figures 5C,D), the top 10 up-regulated and down-regulated genes enriched in immune response signaling are listed in Table 1. Among all altered genes enriched in immune response signaling, IL-18 was found to be the top-ranked gene that was significantly down-regulated in the curcumin group, considering the established profibrotic effect mediated by inflammosome-secreted IL-18 and NLRP3 activation. We speculated that the anti-fibrosis effect exerted by curcumin was mediated by mitigating IL-18 expression and secretion in cardiac fibroblasts.

IL-18 Overexpression Neutralizes Anti-Fibrotic Effect in NRCF Co-cultured With Curcumin-Treated Macrophage
Previously, we identified IL-18 as a central molecule in mediating LPS-treated macrophage-activated myofibroblast transdifferentiation on NRCF, while curcumin administration significantly ameliorated this process. Hence, to test whether compensation of IL-18 in NRCF after co-culture reverses the anti-fibrotic effect of curcumin, we constructed an IL-18 overexpression plasmid and transfected it into TGF-βstimulated NRCF after co-culture with LPS and curcumin double-treated macrophages. We discovered significantly upregulated IL-18 gene expression and considerable transfection efficacy, as demonstrated by RT-PCR and immunofluorescence (Supplementary Figure  S2A,B). Furthermore, IL-18 protein expression levels were determined  Figure S2C). As expected, IL-18 overexpression in NRCF significantly reversed the curcuminmediated anti-fibrotic effect, as shown by the expression of fibrotic genes (i.e., EDA-Fibronectin, Periostin, Vimentin, and α-SMA) were detected by western blot analysis ( Figure 6A-E) more importantly, SMAD2/3 phosphorylation, which was inhibited by curcumin treatment, rebounded by IL-18 overexpression in NRCF ( Figure 6F-H). Furthermore, IL-18 content was also discovered to be significantly down-regulated in plasma from MI + curcumin group compared with MI group using ELISA, which is consistent with the outcome from unbiased transcriptome analysis as well as in vitro findings above ( Figure 6I). From these findings, we conclude that IL-18p-SMAD2/3 signaling plays a critical role in the curcuminmediated anti-fibrosis mechanism in TGF-β-stimulated NRCF co-cultured with LPS-treated macrophages.

DISCUSSION
Cardiac fibrosis, characterized by intersection and collagen deposition within the cardiac interstitium due to net accumulation of extracellular matrix (ECM) proteins, is a common pathophysiological manifestation in most myocardial diseases (Berk et al., 2007) , (Kong et al., 2014). In general, the extent of fibrotic remodeling is closely associated with adverse organ outcomes. Myocardial fibrosis is not necessarily the primary cause of dysfunction. In many circumstances, cardiac fibrosis is the result of a reparative process that is activated in response to cardiomyocyte injury. In humans or other adult mammals, quiescent fibroblasts sustained dynamic balance with cardiomyocytes in healthy status; however, these cells have strong potential in repairing injured myocardium; thus, after pathological conditions, loss of a significant number of cardiomyocytes triggers a reparative program, leading to the formation of fibrous tissue. For example, in acute MI, the sudden death of many cardiomyocytes initiates an intense inflammatory reaction, ultimately leading to the replacement of dead myocardium with a collagen-based scar (Frangogiannis, 2012). Cardiac fibrosis in response to ischemic injury can be divided into three stages: acute early response, proliferation, and late maturation. Inflammation plays a pivotal role in the acute early response, which initiates subsequent repair and acts as a "sentinel." Macrophages are activated immune-cells in early phase post infarction that secrete various cytokines and are intimately correlated with the activation of cardiac fibroblast, initiating "macrophage-fibroblast crosstalk." Proinflammatory activation of cardiac fibroblasts is associated with inflammasome induction, which leads to caspase stimulation and IL-1β secretion by macrophages (Kawaguchi et al., 2011) , (Sandanger et al., 2013). In cultured rat heart fibroblasts, higher gelatinase activity was observed in response to stimulation with IL-1β, and tumor necrosis factor α (TNF-α). Interleukin-1β and tumor necrosis factor alpha decreased collagen synthesis and increased matrix metalloproteinase activity in cardiac fibroblasts in vitro, and they also differentially regulated the production of tissue inhibitors of metalloproteinases. After ischemia reperfusion-induced heart injury, IL-1 receptor 1-deficient mice showed decreased accumulation of macrophages in the infarcted myocardium and diminished early inflammatory and pro-fibrotic responses (Bujak et al., 2007) suggesting downregulation of cardiac repair in a case of disrupted IL-1-dependent signaling. IL-1β inhibits fibroblast proliferation by inducing cell cycle arrest during the G1/S transition (Palmer et al., 1995). However, extended exposure of cardiac fibroblasts to inflammatory cytokines, such as IL-1β, was observed in the prolonged inflammatory phase post-MI. The longterm action of IL-1 β on cardiac fibroblasts delays or prevents the transition from the pro-inflammatory stage to the proliferative phase of heart repair, which may induce adverse remodeling and heart failure by reducing heart contractility and promoting cardiomyocyte apoptosis (Bujak and Frangogiannis, 2009). In contrast to IL-1β, TNF-α may indirectly induce the profibrotic activity of fibroblasts by increasing the expression of type 1 angiotensin II (AT1) receptors. Indeed, most inflammatory cytokines that cooperate in the proinflammatory activation of cardiac fibroblasts can demonstrate effects on fibrotic fibroblast activity. In our study, we revealed that macrophages secreting IL-1β, TNF-α, and IL-6 play a significant role in mediating cardiac fibroblast transdifferentiation, which is in accordance with the conclusions derived from the aforementioned previous studies.
Curcumin has roles in various cardiovascular diseases, including ischemic heart, pressure overload heart, and metabolic disorder-related cardiac diseases. It is well-established that curcumin can directly exerts cardio-protective effect by targeting cardiomyocyte through various of signaling pathway, like disrupts the p300/GATA4 complex and represses agonist-and p300induced hypertrophic responses in cardiomyocytes (Morimoto et al., 2008)or activates the autophagy by upregulating AMPK and JNK1 to alleviate the apoptosis of cardiomyocytes under ischemic stimulation (Yao et al., 2018). However, in our study, we did not confirm the therapeutic effect of curcumin directly in cardiomyocytes, which reflected by the fact that no significant change in cardiomyocyte apoptosis was found in curcumin treated group in mice heart 7 days after MI compared with MI only. In addition, previous studies have also reported that the administration of curcumin in ischemic diseases can salvage the functionality of endothelium (Pu et al., 2013), in our study, we also detected the quantity of CD31 and vWF in ischemic border area but demonstrated no significant difference in the presence or absence of curcumin 28 days after MI (data not shown). The findings illustrated above strongly hints that the regulation of cardiac fibrosis by curcumin may play pivotal role in improving cardiac function after MI. Although it has been well-validated that curcumin inhibits inflammation and anti-ROS as described previously, in a number of studies, curcumin was reported to be directly associated with collagen deposition and fibroblast proliferation. In ischemia-reperfusion (I/R) model, curcumin has been shown to function in regulating both ECM construction and anti-oxidative stress. The downregulated expression of TGF-β1 and p-SMAD2/3 and the upregulation of SMAD7 contributed to this therapeutic effect (Wang et al., 2012). To our surprise, we did not observe obvious inhibition of cardiac fibroblast trans-differentiation via direct treatment of curcumin with NRCF, and phosphorylated SMAD2/3 levels were found to be equal in the presence or absence of curcumin in NRCF, indicating that curcumin only inhibited cardiac fibrosis through macrophages in our study.
The TGF-β1/SMADs signaling pathway has been found to play an important role in inducing and exacerbating the pathological process of myocardial fibrosis after MI (Walton et al., 2017). Specifically, upon binding with the TGF-β1 receptor on the surface of myocardial fibroblasts, TGF-β1 stimulates phosphorylation of downstream SMADs protein (mainly SMAD2/3) and translocation into the nucleus in combination with SMAD4, induces myocardial fibroblast proliferation, phenotypic transformation, and collagen synthesis and ultimately promotes extracellular matrix formation and myocardial fibrosis. In our study, we demonstrated that cardiac fibroblasts received pro-fibrotic cytokines secreted from activated macrophages, thereby enhancing the expression level of IL-18, which in turn promotes phosphorylated SMAD2/3 and subsequent nuclear translocation of p-SMAD2/3, which could be inhibited by curcumin administration on macrophages.