A study on the molecular mechanism of Baicalin in improving Myocardial Fibrosis based on network pharmacology and experimental verification

A study on the molecular mechanism of Baicalin in improving Myocardial Fibrosis based on network pharmacology and experimental verification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A study on the molecular mechanism of Baicalin in improving Myocardial Fibrosis based on network pharmacology and experimental verification Minghao Zhang, Liujun Wu, Yanduo Shen, Jiale Zhang, Bingheng Xie, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4653038/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to evaluate the pharmacological mechanism of baicalin intervention on myocardial fibrosis through network pharmacological analysis, molecular docking, and experimental verification.The chemical components and targets of all the drugs in the Baicalin were obtained through Target Prediction databases. The targets of Myocardial fibrosis(MF)were screened through the GeneCards and OMIM databases. The obtained targets were imported into Cytoscape 3.9 software to construct the active ingredient target network and were imported into the String database to construct PPI network, and the in Cytoscape 3.9 was used for network topology analysis. Gene Ontology (GO) enrichment analysis and Kyoto gene and genomic (KEGG) enrichment analysis were performed on the potential targets of Wuling decoction for MF using the David database.The results were imported into bioinformatics platform to obtain GO and KEGG network relationship maps. The molecular docking software AutoDock Vina was used to dock the core targets with the active ingredients. A MF rat model was established and animals were divided into the control, MF model, a captopril group (9 mg/kg), and low-, middle-, and high-dose baicalin groups (50, 100, 200 mg/kg). Compared with the rats in the MF model group, rats in each administration group demonstrated restoration of ST segment amplitude and T wave on electrocardiograms. Moreover, HWI and LVWI exhibited significant decreases. The levels of CK, LDH, NT-proBNP, Col I and Col III in myocardial tissue also showed significant decreases. Additionally,the degree of myocardial fibrosis was reduced; there were also significant decreases observed in the expression levels of PTGS2 and TNF-α in myocardial tissue, where as an increase was noted in the expression level of IL2. Baicalin has been shown to enhance myocardial fibrosis and cardiac function in a rat model of myocardial fibrosis. The mechanism underlying this effect appears to be associated with the down-regulation of PTGS2 and TNF-αexpression levels, as well as the up-regulation of IL2 expression levels. Baicalin myocardial fibrosis network pharmacology molecular docking experimental verification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Myocardial fibrosis (MF) is a pathological remodeling process that occurs due to the influence of various cardiovascular factors on the myocardial extracellular matrix [ 1 ]. In response to multiple pathological stimuli such as ischemia, hypoxia, and overload, Cardiac fibroblasts (CFs) undergo extensive activation and synthesis of Extracellular matrix (ECM), leading to an imbalance in ECM composition. This results in deposition of type I and type III collagen and fibronectin within the interstitium of cardiomyocytes, ultimately culminating in MF [ 2 ]. Current treatment approaches for MF typically involve cardiotonic, diuretic, vasodilator, and neuroendocrine suppressor medications which can alleviate clinical symptoms; however, these treatments target only specific aspects of the condition and do not effectively reverse the MF process [ 3 ]. The treatment of myelofibrosis (MF) in traditional Chinese medicine has a long history and has shown significant efficacy [ 4 ]. The dried root of Scutellaria baicalensis, a plant in the labiaceae family, is rich in flavonoids, with a particular focus on baicalin. Modern pharmacological research has shown that baicalin possesses strong antioxidant and anti-apoptotic properties. It can also improve myocardial ischemia and restore myocardial function, making it useful in the treatment of cardiovascular diseases [ 5 , 6 ]. However, its potential to improve MF remains uncertain. Therefore, this study initially used network pharmacology and molecular docking methods to predict the potential targets of baicalin for improving MF.Subsequently, captopril was utilized as a positive control drug to observe the effects of baicalin on cardiac function, pathological changes, and fibrosis in MF model rats. This approach confirmed the potential targets of baicalin for improving MF and established a foundation for exploring potential therapeutic drugs for MF (Fig. 1 ). Materials and methods Screen the Potential Targets of Baicalin Used “Baicalin” as keywords to obtain chemical structure of Baicalin through PubChem databases ( https://pubchem.ncbi.nlm.nih.gov/ ), and the potential targets of Baicalin was screened by Swiss Target Prediction databases ( https://www.swisstargetprediction.ch ). Obtaining the Disease Targets Used “Myocardial fibrosis” as keywords to retrieve disease targets through GeneCard databases ( https://www.genecards.org/ ), Online Mendelian Inheritance in Man (OMIM) databases ( https://www.omim.org ) and Therapeutic Target (TTD) Database ( https://db.idrblab.org/ttd ). Construction a Protein-protein Interaction(PPI)Network to Screen out the Key Targets By using Veen Diagram Venny2.1 ( https://bioinfogp.cnb.csic.es/tools/venny/ ), the potential targets and disease targets were crossed with each other and overlapped targets were introduced into String database ( https://cn.string-db.org ), setting the species to be “Homo sapiens,” to obtain protein interaction relations. Then introduced, the outcome was into Cytoscape 3.9.1 software and analyzed nodes and lines using the built-in network analyzer to select the key targets in the PPI network. GO and KEGG Pathway Enrichment Analyses David database ( https://david.ncifcrf.gov ) was used to analyze the KEGG pathway and GO enrichment analysis on the key targets, and the outcome indicated that Baicalin can be used to improve MF through certain signal pathways and potential core targets. The GO enrichment analysis included biological process (BP), cell component (CC) and molecular function (MF). Molecular Docking Verification Baicalin is associated with 3 potential core targets. The compound's structure was determined using the Pubchem database, and the protein structure was determined using the RCSB PBD database ( https://www.rcsb.org ). AutoDock software was utilized for molecular docking, while PyMOL software was employed to optimize the docking process. Animal Grouping, Modeling, and Improvement The MF model was replicated by subcutaneous injection of isoproterenol (Southwest Pharmaceutical Co., Ltd., Chongqing, China) (5 mg/kg) once per day, for 14 days in 53 SD rats (200 ± 20 g; Huaxing experimental animal farm, Zhengzhou, China). After the model was completed, the electrocardiogram was detected. Three rats were randomly selected for anesthesia, and the heart was dissected to observe the pathological changes of myocardial tissue. The model was evaluated by S-T band downshift and fibrosis. 50 successfully modeled rats were randomly divided into model group, captopril group and low-, middle- and high-dose baicalin groups, 10 rats in each group. Another 10 healthy rats were taken as the normal group. According to the literatures, the rats in low-, middle- and high-dose baicalin groups were gavaged with baicalin (Dongguan Jinmeiji Pharmaceutical Co., Ltd., Dongguan, China), with a dose of 50, 100, 200 mg/kg, respectively. The rats in captopril group were gavaged with captopril (Changzhou Pharmaceutical Factory Co., Ltd, Changzhou, China), with a dose of 9 mg/kg [ 7 , 8 ]. The rats in the normal and model group were gavaged with distilled water. The gavage was performed once per day and continued for 28 days. At the 24 hours after the last administration, he rats in each group were anesthetized by intraperitoneal injection of ethyl carbamate (Tianjin Guangfu Technology Development Co, Ltd., Tianjin, China), with a dose of 1 g/kg. The electrocardiogram (ECG) was performed, and the form of S-T band and T wave were used as the index to assess MF. Calculation of HWI and LVWI Heart of rats was dissected and weighed. The body weight (BW), heart weight (HW), and left ventricular weight (LVW) of rats in each group were measured. The HWI (HWI = HW/BW) and LVWI (LVWI = LVW/BW) were calculated to assess the severity of cardiac disease. Determination of myocardium CK,LDH,NT-proBNP,Col I and Col III The frozen myocardial tissue was weighed at 0.1g, and 0.9mL of ice-cold normal saline was added to prepare a 10% homogenate. The homogenate was then centrifuged at 3000 r/min (centrifugation radius 10cm) for 10 minutes, and the supernatant was collected. Following the instructions of the kits (Nanjing Jiancheng Biotechnology Research Institute, Nanjing, China), the absorbance of each sample was determined using Enzyme-linked immunosorbent assay (ELISA). The levels of CK, LDH, NT-proBNP, Col I and Col III were calculated based on the standard curve. Observation of the pathological changes of myocardial tissue Myocardial tissue fixed with paraformaldehyde was obtained and processed to create pathological sections through gradient ethanol dehydration, xylene transparency, paraffin embedding, and 4µm sectioning. Following xylene dewaxing, Hematoxylin-Eosin (HE) staining was conducted on a portion of the sections to observe pathological morphological changes in the cardiac tissue under an optical microscope. The remaining sections were stained with Masson's trichrome stain, resulting in red muscle fibers and blue-green collagen fibers. Subsequently, a random field of vision was selected within each rat myocardial section under the optical microscope. The blue-green positive area and total blue-green positive area within each field were quantified using Image-Pro Plus 6.0 software to calculate the percentage of positive area for evaluating myocardial fibrosis. Determination of myocardium prostaglandin-endoperoxide synthase 2 (PTGS2), tumor necrosis factor (TNF-alpha, TNF-α) and interleukin-2 (IL-2) The tissue sections prepared under item 2.9 were dewaxed and hydrated with xylene, then repaired with citrate buffer for 8 min. After the temperature dropped to room temperature, the sections were incubated with H 2 O 2 for 10 min, soaked with PBS, and incubated with goat serum for 30 min. Then, PTGS2 (1:1000), TNF-α (1:1000), IL-2 (1:1000) antibodies (Servicebio, Co., Ltd., Wuhan, China) were added and incubated at 4 ℃ overnight. The next day, the slices were rewarmed at 37 ℃ for 30 min, followed by HYP-labeled sheep and rabbit antibodies, incubated for 20 min, soaked with PBS, developed with DAB for 10 min, and washed with tap water. Hematoxylin was re-dyed for 3 min, differentiated by hydrochloric alcohol, rinsed with tap water again for bluing, and the slices were dehydrated by ethanol gradient, sealed with transparent xylene and neutral gum for microscopic examination. One field of view was randomly selected in each rat heart tissue section under 400-fold microscope, and brown-yellow color was used as positive staining. Image-Pro Plus 6.0 was used to determine the Integrated Optical Density (IOD) value in each field for statistical analysis. Statistical Analysis The data were analyzed using SPSS 24.0 software (SPSS Inc., Chicago, IL, USA). The means and SDs were compared using a single-factor analysis of variance test with a LSD test. P <0.05 was considered statistically significant. Result Potential Targets of Baicalin 15 potential targets were screened from the Swiss Target Prediction databases (Table 1 ). These targets are possibly involved in improving the metabolic function of Baicalin. Table 1 Potential targets of Baicalin ID Gene Target P15121 AKR1B1 Aldose reductase P30542 ADORA1 Adenosine A1 receptor P01375 TNF TNF-alpha P60568 IL2 Interleukin-2 P47989 XDH Xanthine dehydrogenase P35354 PTGS2 / COX-2 Prostaglandin G/H synthase 2 / Cyclooxygenase-2 P51812 RPS6KA3 Ribosomal protein S6 kinase alpha 3 P00533 EGFR Epidermal growth factor receptor P22303 ACHE Acetylcholinesterase P16083 NQO2 Quinone reductase 2 Q9GZQ4 NMUR2 Neuromedin-U receptor 2 P08913 ADRA2A Alpha-2a adrenergic receptor P18825 ADRA2C Adrenergic receptor alpha-2 Q9NPH5 NOX4 NADPH oxidase 4 P05091 ALDH2 Aldehyde dehydrogenase Disease-associated Targets Used “Myocardial fibrosis” as keywords to retrieve all targets from the GeneCard, OMIM, and TTD databases, then combined the target genes and removed repetitions of 4399 disease targets of MF were found. Analysis of PPI Network and Key Targets of Baicalin in Improving MF 15 potential targets of Baicalin and 4399 targets of MF were inputted into the online visualization platform to construct a Venn diagram and identify thirteen key target genes (Fig. 2). 13 target genes were then entered into the String database to create a PPI network (Fig. 3A), including 13 target proteins and 24 target protein interaction lines, with an average node degree of 3.69 and an average local clustering coefficient of 0.641. The constructed PPI network was imported into Cytoscape3.9 software for visualization processing, with one nonessential target being removed. In descending order of degree value, the key targets of Baicalin for improving MF are TNF-α, PTGS2, EGFR, AKR1B1, XDH, IL2, NOX4, ACHE, ADORA1, ALDH2, ADRA2A, and ADRA2C (Fig. 3B). GO and KEGG Pathway Enrichment Analyses We introduced the 13 key targets into the David database ( P <0.05) for GO enrichment analysis and found that the key targets of Baicalin were mainly enriched within 15 biological processes (BPs), 6 cellular components (CCs), and 7 molecular functions (MFs). The results indicated that the BPs of the key targets of Baicalin in improving MF were mainly related to negative regulation of apoptotic process, positive regulation of MAP kinase activity, and negative regulation of lipid catabolic process, etc.. The analysis of CCs revealed that the identified targets were primarily associated with the extracellular space, integral component of plasma membrane, and basolateral plasma membrane. Additionally, electron carrier activity, protein homodimerization activity, and alpha2-adrenergic receptor activity were all found to be related to the MFs. KEGG pathway analysis ( P <0.05) identified 6 pathways: C-type lectin receptor signaling pathway, Yersinia infection, Alcoholic liver disease, cGMP-PKG signaling pathway, Human cytomegalovirus infection, and Coronavirus disease - COVID-19. This suggests that Baicalin may potentially impact these pathways in treating MF. The bioinformatics platform ( https://www.bioinformatics.com.cn/ ) conducted a visualized analysis of the 10 target genes identified in the GO and KEGG pathway enrichment analysis based on P-value (Fig. 4–5). The target proteins involved in the first C-type lectin receptor signaling pathway (Fig. 6), including TNF-α, PTGS2, and IL2, were all located downstream and were hypothesized to be the core targets of baicalin's efficacy in improving MF. Molecular Docking The molecular docking analysis revealed that Baicalin exhibits strong binding affinity with TNF-α, PTGS2 and IL2, as indicated by the respective binding energies of -7.3, -8.9 and − 8.3 kJ/mol, all of which are lower than − 5 kJ/mol. The results showed that Baicalin forms hydrogen bonds with specific amino acid residues of TNF-α (SER-38, LYS-39, ASN-48, GLU-56 and SER-57), PTGS2 (SER-49, ASP-133, GLY-135, VAL-155, ASP-157 and GLN-327) and IL2 (SER-38, LYS-39, NAS-48, GLU-56, SER57) (Fig. 7). Effects of Baicalin on ECGs in Rats The ECG waveforms of rats in the normal group displayed typical characteristics. In contrast, the model group showed a reduction in the S-T segment of the electrocardiogram, low T wave amplitude, and the presence of atrial flutter. However, both the captopril group and the low-, middle- and high-dose baicalin groups exhibited restoration of S-T segment and T wave amplitude in the electrocardiogram, along with a decrease in atrial flutter (Fig. 8). Effects of Baicalin on HWI and LVWI in Rats Compared to the control group, both HWI and LVWI showed significant increases in the model group, captopril group, and low-, middle-, and high-dose baicalin groups ( P < 0.05). HWI was significantly decreased in the captopril group and middle/high dose baicalin groups compared to the model group ( P < 0.05), while LVWI was significantly decreased in the captopril group and all baicalin dose groups compared to the model group ( P < 0.05). Furthermore, HWI of the low-dose baicalin group was significantly increased compared to the captopril group ( P < 0.05), and LVWI of all baicalin dose groups was significantly increased compared to both the captopril and low-dose baicalin groups ( P < 0.05); Additionally, HWI in middle/high dose baicalin groups was significantly increased compared to the low-dose baicalin group ( P < 0.05) (Fig. 9). Effects of Baicalin on Myocardium CK, LDH and NT-proBNP levels in Rats Compared to the normal group, the levels of CK and LDH in the myocardium were significantly elevated in the model group, captopril group, and low-, middle-, and high dose baicalin groups ( P < 0.05). Additionally, NT-proBNP levels in the myocardium were significantly increased in the model group and low-, middle-, and high dose baicalin groups ( P < 0.05). Furthermore, compared to the model group, levels of CK, LDH and NT-proBNP in myocardial tissue of rats were significantly reduced in the captopril group as well as low-, middle- and high dose baicalin groups ( P < 0.05). Lastly, compared to the captopril group, levels of CK, LDH and NT-proBNP in myocardium were significantly increased in low-, middle-, and high dose baicalin groups ( P < 0.05), while compared with low-dose Baicalein, the levels of CK, LDH and NT-Pro BNP in the myocardial tissue of rats were significantly decreased in the middle- and high-dose groups ( P < 0.05) (Fig. 10). Effects of Baicalin on Myocardium Col Ⅰ and Col Ⅲ levels in Rats The levels of Col Ⅰ and Col Ⅲ in myocardial tissue were significantly increased in the model group, captopril group, and low-, middle-, and high dose baicalin groups compared to the normal group ( P < 0.05). In addition, the levels of Col Ⅰ and Col Ⅲ in myocardium were significantly decreased in the captopril group and low-, middle-, and high dose baicalin groups compared to the model group ( P < 0.05). Furthermore, the levels of Col Ⅰ and Col Ⅲ in myocardium were significantly increased in low- and middle dose baicalin groups compared to the captopril group ( P < 0.05), while they were significantly decreased in middle- and high dose baicalin groups compared to low dose baicalin group ( P < 0.05) (Fig. 11). Effects of Baicalin on Myocardium tissue pathology in Rats The myocardial structure in the normal group appeared to be intact, with clear transverse lines and no evidence of fibrous tissue hyperplasia or inflammatory cell infiltration. In contrast, the model group exhibited myocardial damage, necrosis, hyperplasia of fibrous tissue filling the necrotic space, and inflammatory cell infiltration in the myocardium interstitium. However, in the captopril group and low-, middle- and high-dose baicalin groups, the myocardial structure was orderly with reduced fibrous tissue hyperplasia and no signs of necrosis or inflammatory cell infiltration. Masson staining revealed minimal collagen deposition in the myocardium of the normal group (stained blue), while a large amount was observed in the model group. The degree of collagen deposition was improved to varying degrees in rats from captopril group and low-, middle- and high-dose baicalin groups compared to those from both normal and model groups. Additionally, compared to the model group alone, there was a significant decrease ( P < 0.05) in Masson-staining positive area proportion within each low-, middle-, and high-dose baicalin groups (Fig. 12). Effects of Baicalin on Myocardium TNF-α, PTGS2 and IL2 Protein Expression Levels in Rats The levels of TNF-α and PTGS2 in the myocardium were significantly elevated in the model group, captopril group, and low, medium, and high dose baicalin groups compared to the normal group ( P < 0.05). Conversely, the levels of IL2 in the myocardium were significantly reduced in the model group and low dose baicalin group ( P < 0.05). In comparison to the model group, both TNF-α and PTGS2 levels in the myocardium were significantly decreased in the captopril group and low, medium, and high dose baicalin groups ( P < 0.05), while IL2 levels were significantly increased ( P < 0.05). Furthermore, when compared to the captopril group, TNF-α levels in the myocardium were notably increased in both low and medium dose baicalin groups; PTGS2 levels were also notably increased across all three baicalin dosage groups ( P < 0.05), with a significant decrease observed for IL2 levels specifically within the low dose baicalin group ( P < 0.05). Finally, when comparing with the low dose baicalin group alone,TNF-α levels within rat myocardium from both medium and high-dose groups showed a significant decrease ( P < 0.05), whereas IL2 was found to be markedly increased ( P < 0.05) (Fig. 13). Discussion The pathogenesis of myocardial fibrosis (MF) involves the pathological cross-linking of Col Ⅰ and Col Ⅲ deposition and ECM, leading to changes in mechanical sensory characteristics, hardening of the cavity wall, and impairment of myocardial elasticity as well as systolic and diastolic functions [ 9 ]. Modern medicine emphasizes the close relationship between MF and the regulation of inflammatory cytokines, renin-angiotensin-aldosterone system, chemokines, reactive oxygen species, matrix proteins, and growth factors. Clinical treatment should focus on regulating these physiological processes. While it is challenging to achieve radical treatment for MF, its progression can be delayed to a certain extent [ 10 ]. Traditional Chinese Medicine (TCM) attributes the cause of MF to external sensation, emotion, fatigue with Yang deficiency as the main pathogenesis along with phlegm turbidity, water dampness and blood stasis. The cross-linking involved in MF occurrence can be classified as "toxic evil", thus requiring a treatment principle focused on "removing blood stasis and detoxification" [ 11 ]. Baicalin from scutellaria baicalensis has been selected for this study due to its pharmacodynamic properties including anti-inflammatory effects which are advantageous for cardiomyocyte protection [ 12 ]. Captopril was selected as the positive control in this study because it is a representative Angiotensin converting enzyme (ACE) inhibitor widely used in clinical treatment for MF [ 13 ]. This choice allows for the evaluation of baicalin's potential to improve MF. Isoproterenol, a non-selective β-adrenergic receptor (β-AR) agonist, can activate the β1 receptor of cardiomyocytes. This activation leads to myocardial overexcitability, resulting in strong and sustained contraction of the myocardium and accelerated heart rate. Consequently, ischemic damage of the myocardial tissue may occur. The use of adrenergic receptor (β-AR) can result in an increase in CK, LDH and other biochemical indexes. Sustained myocardial damage can also lead to oxidative stress and inflammation, eventually resulting in MF [ 14 ]. Current studies have shown that the isoproterenol-induced MF model has advantages such as simple operation, short cycle and low technical difficulty which align with the clinical pathological characteristics of MF [ 15 ]. Therefore, this study selected the isoproterenol-induced MF model in rats.ECG, CK and LDH levels are important indicators for clinically evaluating cardiac function; their fluctuation indicates myocardial damage. NT-proBNP is a polypeptide neurohormone secreted by cardiomyocytes; its secretion level is positively correlated with atrial stress tension generated by left ventricular muscle contraction and relaxation -the power source of cardiac ejection-. If left ventricular dysfunction occurs, NT-proBNP levels will increase; thus it's often used as an evaluation index for left ventricular function [ 16 , 17 ] in clinical practice.The results showed that baicalin significantly restored S-T band amplitude and T wave amplitude on electrocardiograms in MF rats while reducing levels of CK, LDH, NT-proBNP Col Ⅰand Col Ⅲ within myocardial tissue. It also improved fibrosis degree within HWI,LVWI,and overall myocardial tissue indicating baicalin's positive effect on improving MF rat conditions. Due to the limited literature reports on the potential enhancement of myocardial fibrosis (MF) by baicalin, further investigation into its specific mechanism is necessary. In this study, network pharmacology and molecular docking techniques were employed to preliminarily predict the core target proteins and pathways involved in baicalin's improvement of MF. The results indicated that the key targets for baicalin in improving MF are TNF-α, PTGS2, and IL2, which are associated with the C-type lectin receptor pathway. The C-type lectin receptor is a superprotein family known for its role in pathogen recognition, signal transduction, and immune response [ 18 ]. However, based on pathway diagram analysis, it is suggested that the three target proteins of TNF-α, PTGS2 and IL2 are effector molecules located downstream of the C-type lectin receptor pathway. Therefore, this study proposes that baicalin may primarily improve MF by regulating the expression levels of TNF-α, PTGS2 and IL2, with little association with the C-type lectin receptor pathway. TNF-α is a multipotent cytokine produced by monocytes, macrophages, lymphocytes and NK cells, which has pro-inflammatory and pro-fibrotic effects. Elevated levels of TNF-α can enhance fibroblast activity and promote collagen synthesis and deposition [ 19 , 20 ]. PTGS2/COX2 is an enzyme that catalyzes the conversion of arachidonic acid into prostaglandins and can be induced by IL-1, TNF-α and other factors. The expression of PTGS2/ Cox2 is normally low in healthy tissues but increases in pathological conditions. Studies have demonstrated increased COX2 expression in ischemic myocardial necrosis induced by subcutaneous injection of isoproterenol; its expression continues to rise as MF worsens [ 21 , 22 ]. IL-2 is secreted by CD4 + T lymphocyte Th1 cells and plays a role in regulating immune function. Currently, it is understood that exogenous IL-2 can enhance the response of regulatory T cells and inhibit fibrosis, while the function of endogenous IL-2 remains unclear [ 23 , 24 ]. The findings of this study revealed an increase in protein levels of TNF-α and PTGS2 in the myocardial tissue of MF rats, accompanied by a decrease in IL-2 protein levels. Following baicalin intervention, there was a decrease in TNF-α and PTGS2 protein levels, as well as an increase in IL-2 protein levels. These results suggest that Baicalin may exert anti-MF effects by down-regulating TNF-α and PTGS2 protein levels while up-regulating IL-2 protein levels in myocardial tissue. Conclusion The present study found Baicalin in the treatment of MF using network pharmacological analysis. TNF-α, PTGS2 and IL2 are the core targets of Baicalin in the treatment of MF. In addition, Baicalin may treat MF patients by regulating signaling pathways such as C-type lectin receptor signaling pathway. In general, this study obtained 3 target genes in the treatment of MF, and pointed out molecular mechanisms of Baicalin for the treatment of MF through network pharmacology and animal experiments verification. Declarations Author contribution Zhang M participated in the design and interpretation of the data and drafting. Xie B, Wu L, and Wu X contribute to data curation and formal analysis. Zhang J and Shen Y performed the collection and assembly of data. Wei D participated in its design and interpretation and helped to revise the manuscript critically. Funding No financial support was allocated for this research. Data Availability Author can confrm that all relevant data are included in the article. The data and materials in the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Ethical Approval This experiment received approval from the Experimental Animal Ethics Committee of Henan University of Chinese Medicine (No. DWLL202301016), and the committee granted permission for the publication of the research results. Informed Consent This article does not contain any studies with human participants performed by any of the authors. Consent for Publication All authors agree to public. Conflict of interest: The authors declare that there is no conflict of interest. References Li Y, Chen H, Cheng M, et al. Research progress of signaling pathway related to iron death and myocardial fibrosis [J]. Journal of Zhengzhou University (Medical Edition), 2024, 59(02): 195-201. Zhang M, Wu X, Wu L, et al. Improvement effect of arbutin on myocardial fibrosis model rats and its mechanism [J]. Chinese Journal of Pharmacy, 2024, 35(5): 529-535. Li C, Fan D. Yang Z, et al. Research progress on the role and mechanism of AMPK in myocardial fibrosis related diseases [J]. Journal of PLA Medicine, 2021, 46(12): 1239-1244. Huo Y, Zhang J, Zhou L, et al. Role of TGF-β/Smads signaling pathway in myocardial fibrosis of heart failure and research status of traditional Chinese medicine intervention [J]. Chinese Journal of Clinical Pharmacology, 2024, 40(3): 444-448. DOI:10.13699/j.cnki.1001-6821.2024.03.029. Xiao Y, Ye J, Zhou Y, Huang J, et al. Baicalin inhibits pressure overload-induced cardiac fibrosis through regulating AMPK/TGF-β/Smads signaling pathway[J]. Arch Biochem Biophys. 2018, 640: 37-46. Liu G, Tao G, Wang H, et al. Effect of baicalin on myocardial hypertrophy and apoptosis induced by abdominal aorta ligation in rats and its mechanism [J]. Journal of Jilin University (Medical Science Edition), 2023, 49(3): 850-857. Wang S, Hu Y, Bao S. Effects of baicalin on ventricular remodeling, ventricular myocyte apoptosis and β1-AR/PKA/CaMKⅡ signaling pathway in rats with dilated cardiomyopathy rats[J]. Chinese Journal of Experimental Formulae, 2018, 24(9): 140-144. Zhang Y, ZHAO S, Ren K, et al. Effect of baicalin on myocardial fibrosis in rats with myocardial infarction [J]. Journal of Cardio-Cerebrovascular Diseases of Integrated Chinese and Western Medicine, 2022, 20(7): 1222-1227. Talman V, Ruskoaho H. Cardiac fibrosis in myocardial infarction-from repair and remodeling to regeneration[J]. Cell Tissue Res. 2016, 365(3):563-581. Liu Y, LIU M, Yang T et al. Advances in the Therapeutic Effect of Chinese Medicine on Myocardial Fibrosis [J]. Pharmacology and Clinic of Traditional Chinese Medicine, 2023, 39 (2): 101-109. Liu J, Zhang H, Tian L, et al. Theoretical research on ‘stasis-toxin’ in myocardial fibrosis of heart failure [J]. Chinese Journal of Traditional Chinese Medicine, 2018,33 (9): 4027-4030. Xu M, Li X, Song L. Baicalin regulates macrophages polarization and alleviates myocardial ischaemia/reperfusion injury via inhibiting JAK/STAT pathway[J]. Pharm Biol. 2020, 58(1): 655-663. Weber KT, Janicki JS, Pick R, et al. Myocardial fibrosis and pathologic hypertrophy in the rat with renovascular hypertension[J]. Am J Cardiol. 1990, 65(14):1G-7G. Pan J, Cao Z, Li N et al. Current situation of common myocardial fibrosis model [J]. Chinese Journal of Clinical Pharmacology, 2021, 37 (1): 84-88. Xu W, Sun Z, Chen H, et al. Comparison of two animal models of myocardial fibrosis [J]. Chinese Journal of Experimental Diagnostics, 2018,22 (4): 708-711. Senni M, Lopez-Sendon J, Cohen-Solal A, et al. Vericiguat and NT-proBNP in patients with heart failure with reduced ejection fraction: analyses from the VICTORIA trial[J]. ESC Heart Fail. 2022, 9(6): 3791-3803. Dirk Von L, Ewald K, Noreert J T, et al. Empagliflozin in acute myocardial infarction: the EMMY trial[J]. Eur Heart J. 2022, 43(41): 4421-4432. Hou H, Guo Y, Chang Q, et al. C-type Lectin Receptor: Old Friend and New Player[J]. Med Chem. 2017, 13(6): 536-543. Verrecchia F, Mauviel A. TGF-beta and TNF-alpha: antagonistic cytokines controlling type I collagen gene expression[J]. Cell Signal. 2004, 16(8): 873-880. Tao L, Xue JF. Effects of TNF-α in rheumatoid arthritis via attenuating α1 (I) collagen promoter[J]. Eur Rev Med Pharmacol Sci. 2018, 22(12): 3905-3912. Xing Z, Yang C, He J, et al. Cardioprotective Effects of Aconite in Isoproterenol-Induced Myocardial Infarction in Rats[J]. 2022, Dec 26: 1090893. Wang J, Lu L, Wang Y, et al. Qishenyiqi Dropping Pill attenuates myocardial fibrosis in rats by inhibiting RAAS-mediated arachidonic acid inflammation[J]. J Ethnopharmacol. 2015, Dec 24: 176: 375-84. Xiao J, Yu K, Li M, et al. The IL-2/Anti-IL-2 Complex Attenuates Cardiac Ischaemia-Reperfusion Injury Through Expansion of Regulatory T Cells[J]. Cell Physiol Biochem. 2017, 44(5): 1810-1827. Yu S, Sun L, Wang H, Autonomic regulation of imbalance-induced myocardial fibrosis and its mechanism in rats with cirrhosis[J]. Exp Ther Med. 2021, 22(3): 1040. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4653038","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":320845313,"identity":"b1b8ea00-e8cd-4c2e-b0e9-94e256d68c7f","order_by":0,"name":"Minghao Zhang","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Minghao","middleName":"","lastName":"Zhang","suffix":""},{"id":320845314,"identity":"02fb84ae-8d2b-49f9-a999-c823fa6cf86e","order_by":1,"name":"Liujun Wu","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Liujun","middleName":"","lastName":"Wu","suffix":""},{"id":320845315,"identity":"8ddac6f3-4e02-498a-a853-83357e47bfa1","order_by":2,"name":"Yanduo Shen","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanduo","middleName":"","lastName":"Shen","suffix":""},{"id":320845316,"identity":"eac71a9c-003a-48d0-9d2c-2dcb52109970","order_by":3,"name":"Jiale Zhang","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiale","middleName":"","lastName":"Zhang","suffix":""},{"id":320845317,"identity":"3e2f8d63-9b76-41fc-b60c-dc85ab2e7ef2","order_by":4,"name":"Bingheng Xie","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bingheng","middleName":"","lastName":"Xie","suffix":""},{"id":320845318,"identity":"ffa4bba1-2b17-4767-aa41-9310ac989824","order_by":5,"name":"Xingfei Wu","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xingfei","middleName":"","lastName":"Wu","suffix":""},{"id":320845319,"identity":"3385f86f-28d2-487f-aa5a-209fb43fa338","order_by":6,"name":"Danan Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACNvbG9h8fKtjq+fmbDxCnhY/ncIPkjDN8CZIzjiUQp0VOwr1BmrdNLsHgQI4BkQ6TYGww5mEzyzM4cObjjTcMdnK6DYS0SDc2JM7hSSuWPNy72XIOQ7Kx2QFCWmQONhx4I3GMse/A2W3SPAwHErcR1CKR2NjAY/CfseFAzjOitTQz8iSwJU44kMNGpBaeg22MMw6wGQMD2dhyjgERfpFvb3/G8PEfmxwwKh/eeFNhJ0dQCwqQ4CEyapC1kKpjFIyCUTAKRgQAAAziRWbhOaDwAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Henan University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Danan","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2024-06-28 07:54:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4653038/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4653038/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60839370,"identity":"588773c7-8fbc-4fcc-bcbb-c50de55bf2cf","added_by":"auto","created_at":"2024-07-22 16:53:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2134195,"visible":true,"origin":"","legend":"\u003cp\u003eThe workfow of elucidating the pharmacological mechanisms of Baicalin in the treatment of myocardial fibrosis based on network pharmacology and experimental verification.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/c13e0ee11f06a713bee35b84.png"},{"id":60839002,"identity":"3689992d-f680-430e-8cba-41507c2f921b","added_by":"auto","created_at":"2024-07-22 16:45:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69503,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/d82b541866f832e71e23fb41.png"},{"id":60839005,"identity":"9a0ce7fd-7ca4-4676-a232-87b791a7d580","added_by":"auto","created_at":"2024-07-22 16:45:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":284119,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure 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legend\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/e5d75d00ee7f68a04be1f9c2.png"},{"id":60839020,"identity":"d1591011-76fe-4e83-9cfd-04ac68415f0f","added_by":"auto","created_at":"2024-07-22 16:45:54","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":106191,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/ba2929af4bdc488c53fa5372.png"},{"id":60839372,"identity":"1fd9f427-9803-4243-8497-3f4e1a31565d","added_by":"auto","created_at":"2024-07-22 16:53:53","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":71482,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/f4a4e913b7e614340160883a.png"},{"id":60839021,"identity":"9a9aa86a-e135-44f7-9e38-96061c0ff496","added_by":"auto","created_at":"2024-07-22 16:45:54","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1599650,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/66b96d1420038e1a3e7d6989.png"},{"id":60839018,"identity":"4139f670-d5c7-41ef-999f-5f41869de440","added_by":"auto","created_at":"2024-07-22 16:45:54","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1243082,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/19b2c41c3dcd6d7a5ca7624a.png"},{"id":91674647,"identity":"f6ca53c7-a4cc-4ae9-a3b9-e334fff9ed45","added_by":"auto","created_at":"2025-09-19 05:01:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8891772,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4653038/v1/a4697056-9065-45a0-8f25-4511443d702c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A study on the molecular mechanism of Baicalin in improving Myocardial Fibrosis based on network pharmacology and experimental verification","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMyocardial fibrosis (MF) is a pathological remodeling process that occurs due to the influence of various cardiovascular factors on the myocardial extracellular matrix [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In response to multiple pathological stimuli such as ischemia, hypoxia, and overload, Cardiac fibroblasts (CFs) undergo extensive activation and synthesis of Extracellular matrix (ECM), leading to an imbalance in ECM composition. This results in deposition of type I and type III collagen and fibronectin within the interstitium of cardiomyocytes, ultimately culminating in MF [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Current treatment approaches for MF typically involve cardiotonic, diuretic, vasodilator, and neuroendocrine suppressor medications which can alleviate clinical symptoms; however, these treatments target only specific aspects of the condition and do not effectively reverse the MF process [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The treatment of myelofibrosis (MF) in traditional Chinese medicine has a long history and has shown significant efficacy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe dried root of Scutellaria baicalensis, a plant in the labiaceae family, is rich in flavonoids, with a particular focus on baicalin. Modern pharmacological research has shown that baicalin possesses strong antioxidant and anti-apoptotic properties. It can also improve myocardial ischemia and restore myocardial function, making it useful in the treatment of cardiovascular diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, its potential to improve MF remains uncertain. Therefore, this study initially used network pharmacology and molecular docking methods to predict the potential targets of baicalin for improving MF.Subsequently, captopril was utilized as a positive control drug to observe the effects of baicalin on cardiac function, pathological changes, and fibrosis in MF model rats. This approach confirmed the potential targets of baicalin for improving MF and established a foundation for exploring potential therapeutic drugs for MF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreen the Potential Targets of Baicalin\u003c/h2\u003e \u003cp\u003eUsed “Baicalin” as keywords to obtain chemical structure of Baicalin through PubChem databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the potential targets of Baicalin was screened by Swiss Target Prediction databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.swisstargetprediction.ch\u003c/span\u003e\u003cspan address=\"https://www.swisstargetprediction.ch\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eObtaining the Disease Targets\u003c/h2\u003e \u003cp\u003eUsed “Myocardial fibrosis” as keywords to retrieve disease targets through GeneCard databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Online Mendelian Inheritance in Man (OMIM) databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omim.org\u003c/span\u003e\u003cspan address=\"https://www.omim.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Therapeutic Target (TTD) Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.org/ttd\u003c/span\u003e\u003cspan address=\"https://db.idrblab.org/ttd\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConstruction a Protein-protein Interaction(PPI)Network to Screen out the Key Targets\u003c/h2\u003e \u003cp\u003eBy using Veen Diagram Venny2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the potential targets and disease targets were crossed with each other and overlapped targets were introduced into String database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), setting the species to be “Homo sapiens,” to obtain protein interaction relations. Then introduced, the outcome was into Cytoscape 3.9.1 software and analyzed nodes and lines using the built-in network analyzer to select the key targets in the PPI network.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGO and KEGG Pathway Enrichment Analyses\u003c/h2\u003e \u003cp\u003eDavid database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the KEGG pathway and GO enrichment analysis on the key targets, and the outcome indicated that Baicalin can be used to improve MF through certain signal pathways and potential core targets. The GO enrichment analysis included biological process (BP), cell component (CC) and molecular function (MF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking Verification\u003c/h2\u003e \u003cp\u003eBaicalin is associated with 3 potential core targets. The compound's structure was determined using the Pubchem database, and the protein structure was determined using the RCSB PBD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). AutoDock software was utilized for molecular docking, while PyMOL software was employed to optimize the docking process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Grouping, Modeling, and Improvement\u003c/h2\u003e \u003cp\u003eThe MF model was replicated by subcutaneous injection of isoproterenol (Southwest Pharmaceutical Co., Ltd., Chongqing, China) (5 mg/kg) once per day, for 14 days in 53 SD rats (200 ± 20 g; Huaxing experimental animal farm, Zhengzhou, China). After the model was completed, the electrocardiogram was detected. Three rats were randomly selected for anesthesia, and the heart was dissected to observe the pathological changes of myocardial tissue. The model was evaluated by S-T band downshift and fibrosis. 50 successfully modeled rats were randomly divided into model group, captopril group and low-, middle- and high-dose baicalin groups, 10 rats in each group. Another 10 healthy rats were taken as the normal group. According to the literatures, the rats in low-, middle- and high-dose baicalin groups were gavaged with baicalin (Dongguan Jinmeiji Pharmaceutical Co., Ltd., Dongguan, China), with a dose of 50, 100, 200 mg/kg, respectively. The rats in captopril group were gavaged with captopril (Changzhou Pharmaceutical Factory Co., Ltd, Changzhou, China), with a dose of 9 mg/kg [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The rats in the normal and model group were gavaged with distilled water. The gavage was performed once per day and continued for 28 days. At the 24 hours after the last administration, he rats in each group were anesthetized by intraperitoneal injection of ethyl carbamate (Tianjin Guangfu Technology Development Co, Ltd., Tianjin, China), with a dose of 1 g/kg. The electrocardiogram (ECG) was performed, and the form of S-T band and T wave were used as the index to assess MF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of HWI and LVWI\u003c/h2\u003e \u003cp\u003eHeart of rats was dissected and weighed. The body weight (BW), heart weight (HW), and left ventricular weight (LVW) of rats in each group were measured. The HWI (HWI = HW/BW) and LVWI (LVWI = LVW/BW) were calculated to assess the severity of cardiac disease.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of myocardium CK,LDH,NT-proBNP,Col I and Col III\u003c/h2\u003e \u003cp\u003eThe frozen myocardial tissue was weighed at 0.1g, and 0.9mL of ice-cold normal saline was added to prepare a 10% homogenate. The homogenate was then centrifuged at 3000 r/min (centrifugation radius 10cm) for 10 minutes, and the supernatant was collected. Following the instructions of the kits (Nanjing Jiancheng Biotechnology Research Institute, Nanjing, China), the absorbance of each sample was determined using Enzyme-linked immunosorbent assay (ELISA). The levels of CK, LDH, NT-proBNP, Col I and Col III were calculated based on the standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eObservation of the pathological changes of myocardial tissue\u003c/h2\u003e \u003cp\u003eMyocardial tissue fixed with paraformaldehyde was obtained and processed to create pathological sections through gradient ethanol dehydration, xylene transparency, paraffin embedding, and 4µm sectioning. Following xylene dewaxing, Hematoxylin-Eosin (HE) staining was conducted on a portion of the sections to observe pathological morphological changes in the cardiac tissue under an optical microscope. The remaining sections were stained with Masson's trichrome stain, resulting in red muscle fibers and blue-green collagen fibers. Subsequently, a random field of vision was selected within each rat myocardial section under the optical microscope. The blue-green positive area and total blue-green positive area within each field were quantified using Image-Pro Plus 6.0 software to calculate the percentage of positive area for evaluating myocardial fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of myocardium prostaglandin-endoperoxide synthase 2 (PTGS2), tumor necrosis factor (TNF-alpha, TNF-α) and interleukin-2 (IL-2)\u003c/h2\u003e \u003cp\u003eThe tissue sections prepared under item 2.9 were dewaxed and hydrated with xylene, then repaired with citrate buffer for 8 min. After the temperature dropped to room temperature, the sections were incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 min, soaked with PBS, and incubated with goat serum for 30 min. Then, PTGS2 (1:1000), TNF-α (1:1000), IL-2 (1:1000) antibodies (Servicebio, Co., Ltd., Wuhan, China) were added and incubated at 4 ℃ overnight. The next day, the slices were rewarmed at 37 ℃ for 30 min, followed by HYP-labeled sheep and rabbit antibodies, incubated for 20 min, soaked with PBS, developed with DAB for 10 min, and washed with tap water. Hematoxylin was re-dyed for 3 min, differentiated by hydrochloric alcohol, rinsed with tap water again for bluing, and the slices were dehydrated by ethanol gradient, sealed with transparent xylene and neutral gum for microscopic examination. One field of view was randomly selected in each rat heart tissue section under 400-fold microscope, and brown-yellow color was used as positive staining. Image-Pro Plus 6.0 was used to determine the Integrated Optical Density (IOD) value in each field for statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using SPSS 24.0 software (SPSS Inc., Chicago, IL, USA). The means and SDs were compared using a single-factor analysis of variance test with a LSD test.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" type=\"Results\" class=\"Section2\"\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003ch2\u003ePotential Targets of Baicalin\u003c/h2\u003e\u003cp\u003e15 potential targets were screened from the Swiss Target Prediction databases (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These targets are possibly involved in improving the metabolic function of Baicalin.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePotential targets of Baicalin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP15121\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAKR1B1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAldose reductase\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP30542\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eADORA1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdenosine A1 receptor\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP01375\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTNF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNF-alpha\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP60568\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eIL2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInterleukin-2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP47989\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eXDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXanthine dehydrogenase\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP35354\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePTGS2 / COX-2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProstaglandin G/H synthase 2 / Cyclooxygenase-2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP51812\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRPS6KA3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRibosomal protein S6 kinase alpha 3\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP00533\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eEGFR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEpidermal growth factor receptor\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP22303\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eACHE\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcetylcholinesterase\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP16083\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNQO2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuinone reductase 2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ9GZQ4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNMUR2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNeuromedin-U receptor 2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP08913\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eADRA2A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAlpha-2a adrenergic receptor\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP18825\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eADRA2C\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdrenergic receptor alpha-2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ9NPH5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNOX4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNADPH oxidase 4\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP05091\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eALDH2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAldehyde dehydrogenase\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eDisease-associated Targets\u003c/h2\u003e\u003cp\u003eUsed “Myocardial fibrosis” as keywords to retrieve all targets from the GeneCard, OMIM, and TTD databases, then combined the target genes and removed repetitions of 4399 disease targets of MF were found.\u003c/p\u003e\u003ch2\u003eAnalysis of PPI Network and Key Targets of Baicalin in Improving MF\u003c/h2\u003e\u003cp\u003e15 potential targets of Baicalin and 4399 targets of MF were inputted into the online visualization platform to construct a Venn diagram and identify thirteen key target genes (Fig.\u0026nbsp;2). 13 target genes were then entered into the String database to create a PPI network (Fig.\u0026nbsp;3A), including 13 target proteins and 24 target protein interaction lines, with an average node degree of 3.69 and an average local clustering coefficient of 0.641. The constructed PPI network was imported into Cytoscape3.9 software for visualization processing, with one nonessential target being removed. In descending order of degree value, the key targets of Baicalin for improving MF are TNF-α, PTGS2, EGFR, AKR1B1, XDH, IL2, NOX4, ACHE, ADORA1, ALDH2, ADRA2A, and ADRA2C (Fig.\u0026nbsp;3B).\u003c/p\u003e\u003ch2\u003eGO and KEGG Pathway Enrichment Analyses\u003c/h2\u003e\u003cp\u003eWe introduced the 13 key targets into the David database (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) for GO enrichment analysis and found that the key targets of Baicalin were mainly enriched within 15 biological processes (BPs), 6 cellular components (CCs), and 7 molecular functions (MFs). The results indicated that the BPs of the key targets of Baicalin in improving MF were mainly related to negative regulation of apoptotic process, positive regulation of MAP kinase activity, and negative regulation of lipid catabolic process, etc.. The analysis of CCs revealed that the identified targets were primarily associated with the extracellular space, integral component of plasma membrane, and basolateral plasma membrane. Additionally, electron carrier activity, protein homodimerization activity, and alpha2-adrenergic receptor activity were all found to be related to the MFs. KEGG pathway analysis (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) identified 6 pathways: C-type lectin receptor signaling pathway, Yersinia infection, Alcoholic liver disease, cGMP-PKG signaling pathway, Human cytomegalovirus infection, and Coronavirus disease - COVID-19. This suggests that Baicalin may potentially impact these pathways in treating MF. The bioinformatics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) conducted a visualized analysis of the 10 target genes identified in the GO and KEGG pathway enrichment analysis based on P-value (Fig.\u0026nbsp;4–5). The target proteins involved in the first C-type lectin receptor signaling pathway (Fig.\u0026nbsp;6), including TNF-α, PTGS2, and IL2, were all located downstream and were hypothesized to be the core targets of baicalin's efficacy in improving MF.\u003c/p\u003e\u003ch2\u003eMolecular Docking\u003c/h2\u003e\u003cp\u003eThe molecular docking analysis revealed that Baicalin exhibits strong binding affinity with TNF-α, PTGS2 and IL2, as indicated by the respective binding energies of -7.3, -8.9 and − 8.3 kJ/mol, all of which are lower than − 5 kJ/mol. The results showed that Baicalin forms hydrogen bonds with specific amino acid residues of TNF-α (SER-38, LYS-39, ASN-48, GLU-56 and SER-57), PTGS2 (SER-49, ASP-133, GLY-135, VAL-155, ASP-157 and GLN-327) and IL2 (SER-38, LYS-39, NAS-48, GLU-56, SER57) (Fig.\u0026nbsp;7).\u003c/p\u003e\u003ch2\u003eEffects of Baicalin on ECGs in Rats\u003c/h2\u003e\u003cp\u003eThe ECG waveforms of rats in the normal group displayed typical characteristics. In contrast, the model group showed a reduction in the S-T segment of the electrocardiogram, low T wave amplitude, and the presence of atrial flutter. However, both the captopril group and the low-, middle- and high-dose baicalin groups exhibited restoration of S-T segment and T wave amplitude in the electrocardiogram, along with a decrease in atrial flutter (Fig.\u0026nbsp;8).\u003c/p\u003e\u003ch2\u003eEffects of Baicalin on HWI and LVWI in Rats\u003c/h2\u003e\u003cp\u003eCompared to the control group, both HWI and LVWI showed significant increases in the model group, captopril group, and low-, middle-, and high-dose baicalin groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). HWI was significantly decreased in the captopril group and middle/high dose baicalin groups compared to the model group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while LVWI was significantly decreased in the captopril group and all baicalin dose groups compared to the model group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Furthermore, HWI of the low-dose baicalin group was significantly increased compared to the captopril group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and LVWI of all baicalin dose groups was significantly increased compared to both the captopril and low-dose baicalin groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05); Additionally, HWI in middle/high dose baicalin groups was significantly increased compared to the low-dose baicalin group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;9).\u003c/p\u003e\u003ch2\u003eEffects of Baicalin on Myocardium CK, LDH and NT-proBNP levels in Rats\u003c/h2\u003e\u003cp\u003eCompared to the normal group, the levels of CK and LDH in the myocardium were significantly elevated in the model group, captopril group, and low-, middle-, and high dose baicalin groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Additionally, NT-proBNP levels in the myocardium were significantly increased in the model group and low-, middle-, and high dose baicalin groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Furthermore, compared to the model group, levels of CK, LDH and NT-proBNP in myocardial tissue of rats were significantly reduced in the captopril group as well as low-, middle- and high dose baicalin groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Lastly, compared to the captopril group, levels of CK, LDH and NT-proBNP in myocardium were significantly increased in low-, middle-, and high dose baicalin groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while compared with low-dose Baicalein, the levels of CK, LDH and NT-Pro BNP in the myocardial tissue of rats were significantly decreased in the middle- and high-dose groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;10).\u003c/p\u003e\u003ch2\u003eEffects of Baicalin on Myocardium Col Ⅰ and Col Ⅲ levels in Rats\u003c/h2\u003e\u003cp\u003eThe levels of Col Ⅰ and Col Ⅲ in myocardial tissue were significantly increased in the model group, captopril group, and low-, middle-, and high dose baicalin groups compared to the normal group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). In addition, the levels of Col Ⅰ and Col Ⅲ in myocardium were significantly decreased in the captopril group and low-, middle-, and high dose baicalin groups compared to the model group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Furthermore, the levels of Col Ⅰ and Col Ⅲ in myocardium were significantly increased in low- and middle dose baicalin groups compared to the captopril group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while they were significantly decreased in middle- and high dose baicalin groups compared to low dose baicalin group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;11).\u003c/p\u003e\u003ch2\u003eEffects of Baicalin on Myocardium tissue pathology in Rats\u003c/h2\u003e\u003cp\u003eThe myocardial structure in the normal group appeared to be intact, with clear transverse lines and no evidence of fibrous tissue hyperplasia or inflammatory cell infiltration. In contrast, the model group exhibited myocardial damage, necrosis, hyperplasia of fibrous tissue filling the necrotic space, and inflammatory cell infiltration in the myocardium interstitium. However, in the captopril group and low-, middle- and high-dose baicalin groups, the myocardial structure was orderly with reduced fibrous tissue hyperplasia and no signs of necrosis or inflammatory cell infiltration. Masson staining revealed minimal collagen deposition in the myocardium of the normal group (stained blue), while a large amount was observed in the model group. The degree of collagen deposition was improved to varying degrees in rats from captopril group and low-, middle- and high-dose baicalin groups compared to those from both normal and model groups. Additionally, compared to the model group alone, there was a significant decrease (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) in Masson-staining positive area proportion within each low-, middle-, and high-dose baicalin groups (Fig.\u0026nbsp;12).\u003c/p\u003e\u003ch2\u003eEffects of Baicalin on Myocardium TNF-α, PTGS2 and IL2 Protein Expression Levels in Rats\u003c/h2\u003e\u003cp\u003eThe levels of TNF-α and PTGS2 in the myocardium were significantly elevated in the model group, captopril group, and low, medium, and high dose baicalin groups compared to the normal group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Conversely, the levels of IL2 in the myocardium were significantly reduced in the model group and low dose baicalin group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). In comparison to the model group, both TNF-α and PTGS2 levels in the myocardium were significantly decreased in the captopril group and low, medium, and high dose baicalin groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while IL2 levels were significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Furthermore, when compared to the captopril group, TNF-α levels in the myocardium were notably increased in both low and medium dose baicalin groups; PTGS2 levels were also notably increased across all three baicalin dosage groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), with a significant decrease observed for IL2 levels specifically within the low dose baicalin group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Finally, when comparing with the low dose baicalin group alone,TNF-α levels within rat myocardium from both medium and high-dose groups showed a significant decrease (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), whereas IL2 was found to be markedly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;13).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pathogenesis of myocardial fibrosis (MF) involves the pathological cross-linking of Col Ⅰ and Col Ⅲ deposition and ECM, leading to changes in mechanical sensory characteristics, hardening of the cavity wall, and impairment of myocardial elasticity as well as systolic and diastolic functions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Modern medicine emphasizes the close relationship between MF and the regulation of inflammatory cytokines, renin-angiotensin-aldosterone system, chemokines, reactive oxygen species, matrix proteins, and growth factors. Clinical treatment should focus on regulating these physiological processes. While it is challenging to achieve radical treatment for MF, its progression can be delayed to a certain extent [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Traditional Chinese Medicine (TCM) attributes the cause of MF to external sensation, emotion, fatigue with Yang deficiency as the main pathogenesis along with phlegm turbidity, water dampness and blood stasis. The cross-linking involved in MF occurrence can be classified as \"toxic evil\", thus requiring a treatment principle focused on \"removing blood stasis and detoxification\" [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Baicalin from scutellaria baicalensis has been selected for this study due to its pharmacodynamic properties including anti-inflammatory effects which are advantageous for cardiomyocyte protection [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Captopril was selected as the positive control in this study because it is a representative Angiotensin converting enzyme (ACE) inhibitor widely used in clinical treatment for MF [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This choice allows for the evaluation of baicalin's potential to improve MF.\u003c/p\u003e\u003cp\u003eIsoproterenol, a non-selective β-adrenergic receptor (β-AR) agonist, can activate the β1 receptor of cardiomyocytes. This activation leads to myocardial overexcitability, resulting in strong and sustained contraction of the myocardium and accelerated heart rate. Consequently, ischemic damage of the myocardial tissue may occur. The use of adrenergic receptor (β-AR) can result in an increase in CK, LDH and other biochemical indexes. Sustained myocardial damage can also lead to oxidative stress and inflammation, eventually resulting in MF [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Current studies have shown that the isoproterenol-induced MF model has advantages such as simple operation, short cycle and low technical difficulty which align with the clinical pathological characteristics of MF [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, this study selected the isoproterenol-induced MF model in rats.ECG, CK and LDH levels are important indicators for clinically evaluating cardiac function; their fluctuation indicates myocardial damage. NT-proBNP is a polypeptide neurohormone secreted by cardiomyocytes; its secretion level is positively correlated with atrial stress tension generated by left ventricular muscle contraction and relaxation -the power source of cardiac ejection-. If left ventricular dysfunction occurs, NT-proBNP levels will increase; thus it's often used as an evaluation index for left ventricular function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] in clinical practice.The results showed that baicalin significantly restored S-T band amplitude and T wave amplitude on electrocardiograms in MF rats while reducing levels of CK, LDH, NT-proBNP Col Ⅰand Col Ⅲ within myocardial tissue. It also improved fibrosis degree within HWI,LVWI,and overall myocardial tissue indicating baicalin's positive effect on improving MF rat conditions.\u003c/p\u003e\u003cp\u003eDue to the limited literature reports on the potential enhancement of myocardial fibrosis (MF) by baicalin, further investigation into its specific mechanism is necessary. In this study, network pharmacology and molecular docking techniques were employed to preliminarily predict the core target proteins and pathways involved in baicalin's improvement of MF. The results indicated that the key targets for baicalin in improving MF are TNF-α, PTGS2, and IL2, which are associated with the C-type lectin receptor pathway. The C-type lectin receptor is a superprotein family known for its role in pathogen recognition, signal transduction, and immune response [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, based on pathway diagram analysis, it is suggested that the three target proteins of TNF-α, PTGS2 and IL2 are effector molecules located downstream of the C-type lectin receptor pathway. Therefore, this study proposes that baicalin may primarily improve MF by regulating the expression levels of TNF-α, PTGS2 and IL2, with little association with the C-type lectin receptor pathway. TNF-α is a multipotent cytokine produced by monocytes, macrophages, lymphocytes and NK cells, which has pro-inflammatory and pro-fibrotic effects. Elevated levels of TNF-α can enhance fibroblast activity and promote collagen synthesis and deposition [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. PTGS2/COX2 is an enzyme that catalyzes the conversion of arachidonic acid into prostaglandins and can be induced by IL-1, TNF-α and other factors. The expression of PTGS2/ Cox2 is normally low in healthy tissues but increases in pathological conditions. Studies have demonstrated increased COX2 expression in ischemic myocardial necrosis induced by subcutaneous injection of isoproterenol; its expression continues to rise as MF worsens [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. IL-2 is secreted by CD4 + T lymphocyte Th1 cells and plays a role in regulating immune function. Currently, it is understood that exogenous IL-2 can enhance the response of regulatory T cells and inhibit fibrosis, while the function of endogenous IL-2 remains unclear [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The findings of this study revealed an increase in protein levels of TNF-α and PTGS2 in the myocardial tissue of MF rats, accompanied by a decrease in IL-2 protein levels. Following baicalin intervention, there was a decrease in TNF-α and PTGS2 protein levels, as well as an increase in IL-2 protein levels. These results suggest that Baicalin may exert anti-MF effects by down-regulating TNF-α and PTGS2 protein levels while up-regulating IL-2 protein levels in myocardial tissue.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study found Baicalin in the treatment of MF using network pharmacological analysis.\u003c/p\u003e \u003cp\u003eTNF-α, PTGS2 and IL2 are the core targets of Baicalin in the treatment of MF. In addition, Baicalin may treat MF patients by regulating signaling pathways such as C-type lectin receptor signaling pathway. In general, this study obtained 3 target genes in the treatment of MF, and pointed out molecular mechanisms of Baicalin for the treatment of MF through network pharmacology and animal experiments verification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution \u0026nbsp;\u003c/strong\u003eZhang M participated in the design and interpretation of the data and drafting. Xie B, Wu L, and Wu X contribute to data curation and formal analysis. Zhang J and Shen Y performed the collection and assembly of data. Wei D participated in its design and interpretation and helped to revise the manuscript critically.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003eNo financial support was allocated for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability \u0026nbsp;\u003c/strong\u003eAuthor can confrm that all relevant data are included in the article. The data and materials in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval \u0026nbsp;\u003c/strong\u003eThis experiment received approval from the Experimental Animal Ethics Committee of Henan University of Chinese Medicine (No. DWLL202301016), and the committee granted permission for the publication of the research results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies with human participants performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication \u0026nbsp;\u003c/strong\u003eAll authors agree to public. Conflict of interest: The authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi Y, Chen H, Cheng M, et al. Research progress of signaling pathway related to iron death and myocardial fibrosis [J]. Journal of Zhengzhou University (Medical Edition), 2024, 59(02): 195-201. \u003c/li\u003e\n\u003cli\u003eZhang M, Wu X, Wu L, et al. Improvement effect of arbutin on myocardial fibrosis model rats and its mechanism [J]. Chinese Journal of Pharmacy, 2024, 35(5): 529-535.\u003c/li\u003e\n\u003cli\u003eLi C, Fan D. Yang Z, et al. Research progress on the role and mechanism of AMPK in myocardial fibrosis related diseases [J]. Journal of PLA Medicine, 2021, 46(12): 1239-1244.\u003c/li\u003e\n\u003cli\u003eHuo Y, Zhang J, Zhou L, et al. Role of TGF-\u0026beta;/Smads signaling pathway in myocardial fibrosis of heart failure and research status of traditional Chinese medicine intervention [J]. Chinese Journal of Clinical Pharmacology, 2024, 40(3): 444-448. DOI:10.13699/j.cnki.1001-6821.2024.03.029.\u003c/li\u003e\n\u003cli\u003eXiao Y, Ye J, Zhou Y, Huang J, et al. Baicalin inhibits pressure overload-induced cardiac fibrosis through regulating AMPK/TGF-\u0026beta;/Smads signaling pathway[J]. Arch Biochem Biophys. 2018, 640: 37-46.\u003c/li\u003e\n\u003cli\u003eLiu G, Tao G, Wang H, et al. Effect of baicalin on myocardial hypertrophy and apoptosis induced by abdominal aorta ligation in rats and its mechanism [J]. Journal of Jilin University (Medical Science Edition), 2023, 49(3): 850-857.\u003c/li\u003e\n\u003cli\u003eWang S, Hu Y, Bao S. Effects of baicalin on ventricular remodeling, ventricular myocyte apoptosis and \u0026beta;1-AR/PKA/CaMKⅡ signaling pathway in rats with dilated cardiomyopathy rats[J]. Chinese Journal of Experimental Formulae, 2018, 24(9): 140-144.\u003c/li\u003e\n\u003cli\u003eZhang Y, ZHAO S, Ren K, et al. Effect of baicalin on myocardial fibrosis in rats with myocardial infarction [J]. Journal of Cardio-Cerebrovascular Diseases of Integrated Chinese and Western Medicine, 2022, 20(7): 1222-1227.\u003c/li\u003e\n\u003cli\u003eTalman V, Ruskoaho H. Cardiac fibrosis in myocardial infarction-from repair and remodeling to regeneration[J]. Cell Tissue Res. 2016, 365(3):563-581.\u003c/li\u003e\n\u003cli\u003eLiu Y, LIU M, Yang T et al. Advances in the Therapeutic Effect of Chinese Medicine on Myocardial Fibrosis [J]. Pharmacology and Clinic of Traditional Chinese Medicine, 2023, 39 (2): 101-109.\u003c/li\u003e\n\u003cli\u003eLiu J, Zhang H, Tian L, et al. Theoretical research on \u0026lsquo;stasis-toxin\u0026rsquo; in myocardial fibrosis of heart failure [J]. Chinese Journal of Traditional Chinese Medicine, 2018,33 (9): 4027-4030.\u003c/li\u003e\n\u003cli\u003eXu M, Li X, Song L. Baicalin regulates macrophages polarization and alleviates myocardial ischaemia/reperfusion injury via inhibiting JAK/STAT pathway[J]. Pharm Biol. 2020, 58(1): 655-663.\u003c/li\u003e\n\u003cli\u003eWeber KT, Janicki JS, Pick R, et al. Myocardial fibrosis and pathologic hypertrophy in the rat with renovascular hypertension[J]. Am J Cardiol. 1990, 65(14):1G-7G.\u003c/li\u003e\n\u003cli\u003ePan J, Cao Z, Li N et al. Current situation of common myocardial fibrosis model [J]. Chinese Journal of Clinical Pharmacology, 2021, 37 (1): 84-88.\u003c/li\u003e\n\u003cli\u003eXu W, Sun Z, Chen H, et al. Comparison of two animal models of myocardial fibrosis [J]. Chinese Journal of Experimental Diagnostics, 2018,22 (4): 708-711.\u003c/li\u003e\n\u003cli\u003eSenni M, Lopez-Sendon J, Cohen-Solal A, et al. Vericiguat and NT-proBNP in patients with heart failure with reduced ejection fraction: analyses from the VICTORIA trial[J]. ESC Heart Fail. 2022, 9(6): 3791-3803.\u003c/li\u003e\n\u003cli\u003eDirk Von L, Ewald K, Noreert J T, et al. Empagliflozin in acute myocardial infarction: the EMMY trial[J]. Eur Heart J. 2022, 43(41): 4421-4432.\u003c/li\u003e\n\u003cli\u003eHou H, Guo Y, Chang Q, et al. C-type Lectin Receptor: Old Friend and New Player[J]. Med Chem. 2017, 13(6): 536-543.\u003c/li\u003e\n\u003cli\u003eVerrecchia F, Mauviel A. TGF-beta and TNF-alpha: antagonistic cytokines controlling type I collagen gene expression[J]. Cell Signal. 2004, 16(8): 873-880.\u003c/li\u003e\n\u003cli\u003eTao L, Xue JF. Effects of TNF-\u0026alpha; in rheumatoid arthritis via attenuating \u0026alpha;1 (I) collagen promoter[J]. Eur Rev Med Pharmacol Sci. 2018, 22(12): 3905-3912.\u003c/li\u003e\n\u003cli\u003eXing Z, Yang C, He J, et al. Cardioprotective Effects of Aconite in Isoproterenol-Induced Myocardial Infarction in Rats[J]. 2022, Dec 26: 1090893.\u003c/li\u003e\n\u003cli\u003eWang J, Lu L, Wang Y, et al. Qishenyiqi Dropping Pill attenuates myocardial fibrosis in rats by inhibiting RAAS-mediated arachidonic acid inflammation[J]. J Ethnopharmacol. 2015, Dec 24: 176: 375-84.\u003c/li\u003e\n\u003cli\u003eXiao J, Yu K, Li M, et al. The IL-2/Anti-IL-2 Complex Attenuates Cardiac Ischaemia-Reperfusion Injury Through Expansion of Regulatory T Cells[J]. Cell Physiol Biochem. 2017, 44(5): 1810-1827.\u003c/li\u003e\n\u003cli\u003eYu S, Sun L, Wang H, Autonomic regulation of imbalance-induced myocardial fibrosis and its mechanism in rats with cirrhosis[J]. Exp Ther Med. 2021, 22(3): 1040.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Baicalin, myocardial fibrosis, network pharmacology, molecular docking, experimental verification","lastPublishedDoi":"10.21203/rs.3.rs-4653038/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4653038/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to evaluate the pharmacological mechanism of baicalin intervention on myocardial fibrosis through network pharmacological analysis, molecular docking, and experimental verification.The chemical components and targets of all the drugs in the Baicalin were obtained through Target Prediction databases. The targets of Myocardial fibrosis(MF)were screened through the GeneCards and OMIM databases. The obtained targets were imported into Cytoscape 3.9 software to construct the active ingredient target network and were imported into the String database to construct PPI network, and the in Cytoscape 3.9 was used for network topology analysis. Gene Ontology (GO) enrichment analysis and Kyoto gene and genomic (KEGG) enrichment analysis were performed on the potential targets of Wuling decoction for MF using the David database.The results were imported into bioinformatics platform to obtain GO and KEGG network relationship maps. The molecular docking software AutoDock Vina was used to dock the core targets with the active ingredients. A MF rat model was established and animals were divided into the control, MF model, a captopril group (9 mg/kg), and low-, middle-, and high-dose baicalin groups (50, 100, 200 mg/kg). Compared with the rats in the MF model group, rats in each administration group demonstrated restoration of ST segment amplitude and T wave on electrocardiograms. Moreover, HWI and LVWI exhibited significant decreases. The levels of CK, LDH, NT-proBNP, Col I and Col III in myocardial tissue also showed significant decreases. Additionally,the degree of myocardial fibrosis was reduced; there were also significant decreases observed in the expression levels of PTGS2 and TNF-α in myocardial tissue, where as an increase was noted in the expression level of IL2. Baicalin has been shown to enhance myocardial fibrosis and cardiac function in a rat model of myocardial fibrosis. The mechanism underlying this effect appears to be associated with the down-regulation of PTGS2 and TNF-αexpression levels, as well as the up-regulation of IL2 expression levels.\u003c/p\u003e","manuscriptTitle":"A study on the molecular mechanism of Baicalin in improving Myocardial Fibrosis based on network pharmacology and experimental verification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 16:45:49","doi":"10.21203/rs.3.rs-4653038/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b92eb73-aecf-43d0-bdd8-58365d73b51b","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-19T04:53:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 16:45:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4653038","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4653038","identity":"rs-4653038","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}