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Metabolic syndrome (MetS), the presence of at least three of five manifestations, obesity, insulin resistance, dyslipidemia, and hypertension, has been considered a major risk factor for cardiovascular diseases (CVD) such as cardiac fibrosis. ^{1 – 3} The risk of cardiovascular disease was seven times higher among individuals with MetS and diagnosed diabetes mellitus. ⁴ Overnutrition and lack of physical activities leading to obesity and diabetes presented as the most common etiology of MetS. ⁵

Obesity and diabetes caused reactive oxygen species (ROS) production that activated neurohumoral systems, including the renin-angiotensin system (RAS). ROS and RAS stimulated inflammation through inflammation mediators release and immune cell activation. ⁶ Angiotensin-II and TGFβ-1, well known potent pro-fibrotic agents, lead to fibroblast differentiation and excessive ECM production. ^{7 , 8} Previous studies suggested that rats or mice with a high-fat diet and low-dose streptozotocin showed cardiac morphology alteration with evidence of inflammation and oxidative stress, leading to cardiac fibrosis. ^{9 , 10} It showed cardiac fibrosis features characterized by functional and structural cardiac changes through morphological, biochemical, and molecular alterations. ¹¹

Treatment strategies for MetS and its complication mostly used conventional drugs to solve one or some metabolic manifestation, but it failed due to its inability to reduce the cardiovascular complications especially heart damage. ^{12 – 16} Recently, scientists have focused on bioactive phytochemicals due to their wide range of pharmacological activities. Epigallocatechin-gallate (EGCG), a bioactive phenolic from green tea leaves, is well known for its antioxidant and anti-inflammatory effects. ¹⁷ Besides that, chlorogenic acid (CGA), a phenolic compound from plants such as green coffee beans, is also known for its antioxidant and anti-inflammation activities. ¹⁸ Combining two antioxidants significantly attenuates MetS metabolic parameters rather than individual substrate administration, clinically and molecularly. ^{19 , 20} Furthermore, EGCG signifies the internal mechanism of the anti-fibrosis effect by preventing NF-κB transfer to the nucleus and downregulating CTGF gene expression that reduces fibroblast proliferation and synthesis of extracellular matrix. ²¹ Moreover, CGA from coffee administration improved the outcomes of diabetes and non-alcoholic fatty liver disease (NAFLD) patients through a TGF-β1 lowering effect, ²² and worked as an anti-fibrosis effect on the heart. ²³ Nevertheless, previous studies reported a drawback of green coffee administration to CVD risk due to its caffeine content. ^{24 – 26} Therefore, this study investigated the administration of green tea and decaffeinated light roasted green coffee extract combination on prevention of cardiac MetS complications, especially cardiac fibrosis, through inhibition of inflammation responses.

MetS remains a major risk factor for CVD and global health concerns. ²⁸ The mechanism underlies MetS are excessive ROS production and low-grade chronic inflammation. ^{29 – 31} GT and GC extract both has antioxidant and anti-inflammatory effect with their major bioactive compounds, EGCG and CGA, which is proven for various disease treatments ^{17 , 32 – 37} though the possible role of their combination extract against cardiac fibrosis induced by MetS remains to be elucidated. In this study, HFHS/LDSTZ-MetS rat model was treated with 300 mg/kg b.w. GT extract, 200 mg/kg b.w. GC extract and combination of those two extracts (300 mg/kg b.w. green tea extract and 200 mg/kg b.w. decaffeinated light roasted green coffee extract). Metabolic parameters of those extracts treated animals were already described in Rohman et al. (2021). ²⁰ The data revealed attenuation of body weight, fasting blood glucose level, HDL, LDL, total cholesterol level, and systolic blood pressure in the MetS rat model. Administration of a combination of GT and GC extracts showed better alleviation of metabolic parameters than that of single extract administration. ²⁰ This study is the first report that showed the effect of GT and GC extracts combination dose against MetS induced cardiac fibrosis through inflammation alleviation.

This study showed that rats with HFHS/LD-STZ-induced MetS increased cardiac fibrosis biomarkers by increasing angiotensin-2 serum and cardiac inflammation gene expression levels. A previous study also denoted cardiac fibrosis by increased cardiac galectin-3 mRNA and collagen deposition. ¹⁰ High-fat and high-sucrose diet and low-dose STZ induction causes oxidative stress in many tissues, such as adipose, liver, pancreas, heart, and skeletal muscle. ³⁰ Increases in ROS production stimulate cells to produce angiotensinogen then converted to angiotensin-2 (Ang-2) throughout the body. ^{8 , 38 – 40} Furthermore, ROS can activate signaling kinases and transcription factors such as Rac GTP-binding protein, associated with fibrosis and hypertrophy, and the NF-κB pathway, related to pro-inflammatory gene transcription. ⁴¹ Moreover, visceral obesity is strongly correlated with epicardial adipose tissue, and it means a more significant amount of Ang-2 and pro-inflammatory adipokines production. ⁴² Ang-2 also stimulates expression and activates NF-kB to promote more inflammation. ⁸ All the induction and inflammation processes continuously develop, causing chronic inflammation and promoting cardiac fibrosis development.

Ang-2 serum levels were significantly lower in the GT, GC, and CE group than in the MetS group. The most significant lower level of Ang-2 was observed in the CE dose. Earlier data showed that TNF-α serum level was decreased most at combination doses of 300 mg/kg b.w. GT extract and 200 mg/kg b.w. GC extract. ²⁰ Ang-2 can express and activate NF-kB to promote inflammation with the release of TNF-α, IL-6, and TGF-β1, so reducing of its serum level impacts NF-kB and inflammatory mediators. ^{8 , 43 – 46} Interleukin-6 denoted its role in collagen synthesis through STAT-3 signaling activation in cardiac fibroblast. ⁴⁷ Moreover, overexpression of cardiac TNF-α caused cardiac fibrosis with heart failure development by involved collagen synthesis and activating matrix-degrading proteases (MMP2 and MMP9), which activates latent TGF-β1. ⁴⁸ In this present study, NFkB, TNF-α, and IL-6 relative mRNA expression level in the heart of the metabolic syndrome rat model treated with GT and GC extracts showed a lower level compared to that of MetS group. The lowest inflammatory markers were observed in the CE groups. It suggested that a combination of GT and GC extract worked better in lowering inflammation.

TGF-β1 is a potent pro-fibrotic factor affected by increased Ang-2, inflammation mediators, and NF-kB. Lu et al. (2021) reported a rise of TGF-β1, α-SMA, and Col-1 relative mRNA levels on heart tissue and cardiac fibrotic histopathological finding in high fat and low-dose STZ-induced diabetic mice. ⁹ Rac1 is a small GTPase molecule, one of TGF-β1 noncanonical signaling pathway molecules that function on the regulation of expression and activation of the actin cytoskeleton, including α-SMA as actin stress fiber myofibroblast. ^{49 – 53} α-SMA is a hallmark marker of fibrosis that indicates fibroblast differentiation to mature myofibroblast. It can stretch more, attracting ECM and cells in the cardiac tissue environment that causes interstitial fibrosis and stiffness. ^{54 , 55} The relative mRNA expression level of TGF-β1, Rac1, and α-SMA in the heart decreased in all extracts, but the most significant decrease was at combination dose. This study suggested that a combination of GT and GC extracts attenuated MetS-induced cardiac fibrosis better than the administration of a single extract. Nevertheless, Rac1 also plays a role in GLUT4 translocation in skeletal muscle by activating and modulating actin cytoskeleton to transfer GLUT4 vesicles from the cytoplasm to the membrane. ^{56 – 58} It was contradictory with our recent study that the lower relative expression of Rac1 in the heart did not affect the fasting blood glucose. A possible explanation of this issue was that Rac1 was not involved in GLUT4 translocation signaling pathways in the rat heart. Hence, further study is needed to investigate GLUT4 mRNA expression level in rat hearts as well as the protein level of molecules involved in this pathway.

This study suggested that the HFHS/LDSTZ-MetS model showed sustained cardiac fibrosis through enhancement of RAS and inflammation. However, GT and GC extract were attenuated cardiac fibrosis markers with the most optimal dose at the combination of 300 mg/kg b.w. GT extract and 200 mg/kg b.w. GC extract. The combination administration lowered Ang-II serum level and relative mRNA expression level of NFkB, TNF-α, IL-6, TGF-β1, Rac1, and α-SMA on heart rat tissue. All these results provided a clear explanation regarding the effect of combination GT and GC extracts against MetS-induced cardiac fibrosis via inflammation inhibition.

Figshare: Underlying data for ‘Improvement of cardiac fibrosis biomarkers through inflammation inhibition by green tea and decaffeinated light roasted green coffee extract combination administration in metabolic syndrome rat model’ https://doi.org/10.6084/m9.figshare.13249163.v3

This project contains the following underlying data: -

Data of Metabolic Syndrome Rat Model.xlsx (This file contains the analyzed data)

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

D.A.N: Conceptualization, Data Curation, Methodology, Resources

M.L: Conceptualization, Data Curation, Methodology, Writing – Review & Editing

M.S.R: Conceptualization, Funding acquisition, Supervision, Writing – Review & Editing

N.A.W: Data Curation, Investigation, Writing – Original Draft Preparation

M.N.R: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation

N.W: Project Administration, Supervision, Writing – Review & Editing