Mechanisms of traditional Chinese medicine in modulating cardiac microvascular endothelial cells in various injury models: A comprehensive systematic review

1 / 58 1 Mechanisms of traditional Chinese medicine in 2 modulating cardiac microvascular endothelial 3 cells in various injury models: A comprehensive 4 systematic review 5 6 7 Huiwen Zhou1,2, Hongxu Liu1*, Xiang Li1,3*, Juju Shang1, Jiaping Chen1,2, Huiqi Zong1,4 8 9 10 11 1. Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, 12 China 13 2. Capital Medical University, Beijing, China 14 3. Lhasa people’s hospital, Lhasa, China 15 4. Beijing University of Chinese Medicine, Beijing, China 16 17 18 *Corresponding author: 19 E-mail: [email protected] (HL); [email protected] (XL) 20 21 22 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 2 / 58 23 Abstract 24 Background 25 The structural and functional failure of cardiac microvascular endothelial cells (CMECs) is 26 a primary contributor to coronary microvascular dysfunction (CMD). Traditional Chinese 27 medicine (TCM) has been identified as a potential therapeutic approach for preserving 28 CMECs and mitigating CMD. 29 Objective 30 This systematic review aims to present the latest evidence on TCM intervention 31 mechanisms in CMECs under diverse injury models. 32 Methods 33 This systematic review was performed following the parameters of the PRISMA statement 34 (Preferred Reporting Items for Systematic Reviews and Meta-Analysis). A comprehensive 35 literature search was conducted using PubMed, Embase, Web of Science, Scopus, China 36 National Knowledge Infrastructure and China Biology Medicine disc. Reference lists of 37 selected articles were reviewed to identify relevant studies. The search was not limited by 38 year and was conducted solely in English. Eligible studies comprised publications 39 describing in vitro studies that presented the latest evidence on TCM intervention 40 mechanisms in CMECs under diverse injury models. 41 Results 42 A total of 63 papers were included in this study. According to the cell processing approach, 43 19 studies on ischemia or hypoxic injury models, 16 studies on Ischemia/reperfusion (I/R) 44 or hypoxia/reoxygenation (H/R) injury models, 10 studies on inflammatory injury models, 5 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 3 / 58 45 studies on metabolic injury models, 3 studies on angiotensin II injury models, and 10 46 studies on other models. TCM exhibits structural and functional intervention capabilities in 47 diverse damage conditions of CMECs. Its mechanism of action involves antioxidant, anti- 48 apoptotic, anti-inflammatory effects, as well as regulation of energy metabolism through 49 signaling pathways such as HIF-1α/VEGF, PI3K/AKT, MAPK, and NF-κB. 50 Conclusions 51 The CCM and its constituents modulate CMECs through multiple signaling pathways in 52 response to various injury models, thereby conferring protection on the coronary 53 microcirculation. 54 Keywords 55 Traditional Chinese medicine, Cardiovascular disease, Cardiac microvascular endothelial 56 cells, Coronary microvascular dysfunction, Mechanisms 57 58 Abbreviations: 4-ACGC, 4-O-(2-O-acetyl-6-O-p-coumaroyl-β-D-glucopyranosyl)-p- 59 coumaric acid; A, Allicin; ADM, Adriamycin; AJs, Adherens junctions; AKT, Protein kinase 60 B; ALDH, Aldehyde dehydrogenase; AMPK, Adenosine 5′-monophosphate activated 61 protein kinase; AND, Andrographolide; Ang Ⅱ, Angiotensin II; Angptl4, Angiopoietin-like 62 4; AP, Astragalus polysaccharide; ApoE, Apolipoprotein E; ARE, Antioxidant response 63 element; ASIV, Astragaloside IV; ATP, Adenosine triphosphate; ATM, Ataxia 64 telangiectasia mutated; bFGF, Basic fibroblast growth factor; BTE, Black tea extract; CBF, 65 Coronary blood flow; CCM, Compound Chinese medicine; CFs, Cardiac fibroblasts; CFR, 66 Coronary flow reserve; CHM, Chinese herbal medicines; CL, Carthamus tinctorius L. and .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 4 / 58 67 Lepidium apetalum Willd; CMD, Coronary microvascular dysfunction; CMVD, Coronary 68 microvascular disease; CMECs, Cardiac microvascular endothelial cells; COX-2, 69 Cyclooxygenase-2; CTSS, Cathepsin S; CVB3, Coxsackievirus B3; CYP, Alpha-Cyperone; 70 CYT, Caffeoylxanthiazonoside; DG, GeGen DanShen extract; DGBX, DangGuiBuXue 71 Tang; Diosmetin-7-O-glucoside, Diosmetin-7-O-β-D-glucopyranoside; DLF, DanLou 72 formula; Drp1, Dynamin-related protein 1; ECE-1, Endothelin-converting enzyme-1; 73 EGb761, Ginkgo biloba extract; EndMT, Endothelial-to-mesenchymal transition; eNOS, 74 Endothelial nitric oxide synthase; ER, Estrogen receptor; ERK, Extracellular signal- 75 regulated kinase; ERS, Endoplasmic reticulum stress; FGF, Fibroblast growth factor; FoxO, 76 Forkhead-Box Class O; GAS, Gastrodin; GTP, Guanosine triphosphate; H3K9ac, Histone 77 H3 lysine 9 acetylation; H/R, Hypoxia/Reoxygenation; Hcy, Homocysteine; HF, Heart 78 failure; HFD, High fat diet; HG, High glucose; HIF, Hypoxia-inducible factor; HO-1, Heme 79 oxygenase-1; HSP27, Heat shock protein 27; I/R, Ischemia/reperfusion; IκB, Inhibitor of 80 NF-κB; IGF1R, Insulin-like growth factor 1 receptor; IHD, Ischemic heart disease; IL, 81 Interleukin; iNOS, Inducible nitric oxide synthase; IRS1, Insulin receptor substrate 1; JAK, 82 Janus tyrosine kinase; JAM, Junctional adhesion molecule; KLF, Krüppel-like factor; L- 83 NAME, N(ω)-nitro-L-arginine-methyl ester; LDL-C, Low-density lipoprotein cholesterol; 84 Linc-ROR, Long intergenic non-coding RNA regulator of reprogramming; LPS, 85 Lipopolysaccharide; MAPK, Mitogen-activated protein kinase; MBC, Major bioactive 86 component; MDA, Malondialdehyde; MDG-1, A water-soluble beta-D-fructan from O. 87 japonicus; MEK, Mitogen-activated protein kinase; MF, Myocardial fibrosis; Mff, 88 mitochondrial fission factor; MHBFC, 17-Methoxyl-7-hydroxy-benzene-furanchalcone; MI, .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 5 / 58 89 Myocardial infarction; MLC, Myosin light chain; MMP-9, Matrix metallopeptidase-9; mTOR, 90 Mammalian target of rapamycin; NADPH, Nicotinamide adenine dinucleotide phosphate; 91 NF-κB, Nuclear factor-kappa B; NLRP3, Nucleotide-binding oligomerization domain-like 92 receptor protein 3; NO, Nitric oxide; NOS, Nitric oxide synthase; NR4A1, Nuclear receptor 93 subfamily 4 group A member 1; Nrf2, Nuclear factor‑erythroid 2 related factor 2; NRG-1, 94 Neuregulin-1; NT, Nitrotyrosine; OA, Oroxylin A; OJ, Ophiopogon japonicus; P-IκBα, 95 Phosphorylated inhibitor of kappa B alpha; P-p65, Phosphorylated p65; P70S6k1, 70 kDa 96 ribosomal protein S6 kinase 1; PAI, Plasminogen activator inhibitor; PDGF, Platelet- 97 derived growth factor; PECAM-1, Platelet-endothelial cell adhesion molecule-1; PER, 98 Periplocin; PGE2, Prostaglandin E2; PGIS, Prostacyclin synthase; PHDs, Prolyl 99 hydroxylase domain enzymes; PI3K, Phosphatidylinositol 3-kinase; PINK, Phosphatase 100 and tensin homolog-induced putative kinase; PLCδ3, Phospholipase Cδ3; PMN, 101 Polymorphonuclear leukocyte; PN, Panax Notoginseng; PPAR-α, Peroxisome proliferator- 102 activated receptor α; PRISMA, Preferred Reporting Items for Systematic Reviews and 103 Meta-Analyses; Pyk, Proline-rich tyrosine kinase; Q, Quercetin; QLQX, QiLiQiangXin; 104 QSYQ, QiShenYiQi; Rb3, Ginsenoside-Rb3; RG, Radix Ginseng; Rg3, Ginsenoside-Rg3; 105 RhoA, Ras homolog gene family member A; RIP3, Receptor interacting protein 3; RLC, 106 Rhizoma Ligustici Chuanxiong ; ROCK, Rho-associated protein kinase; ROS, Reactive 107 oxygen species; RPS6KB1, Ribosomal protein S6 kinase B1; RS, Radix Scutellariae; RSV, 108 Resveratrol; S1P, Sphingosine 1 phosphate; SA, Salidroside; SCU, Scutellarin; SL, 109 ShenLian extract; SMF, ShenMai formula; SOD, Superoxide dismutase; SSNX, 110 ShuangShenNingXin formula; STAT, Signal transducer and activator of transcription; .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 6 / 58 111 STDP, SheXiangTongXin Dropping Pill; SXBX, SheXiangBaoXin Pill; Tan Ⅱ A, 112 Tanshinone Ⅱ A; TCM, Traditional Chinese medicine; TF, Tissue factor ; TGF, 113 Transforming growth factor; TJs, Tight junctions; TMYX, TongMaiYangXin pill; TNF-α, 114 Tumor necrosis factor-α; TXD, TianXiangDan; TXL, TongXinLuo; VA, Velvet Antler; VAP, 115 Velvet antler proteins; VE, Vascular endothelial; VEGF, Vascular endothelial growth factor; 116 VMC, Viral myocarditis; VSMCs, Vascular smooth muscle cells; XQT, XiangQiTang; ZO- 117 1, Zona occludens-1. 118 119 1. Introduction 120 Coronary microvascular disease (CMVD) is a clinical condition characterized by 121 objective evidence of exertional angina and/or myocardial ischemia resulting from 122 structural and/or functional abnormalities in the coronary microcirculation, triggered by 123 various etiological factors. Coronary microvascular dysfunction (CMD) represents a crucial 124 mechanism underlying CMVD [1]. CMD is implicated in different stages of cardiovascular 125 disease and serves as a significant adverse prognostic factor for patients with ischemic 126 heart disease (IHD) [2]. In recent years, CMD has garnered increasing attention due to its 127 clinical relevance. Notwithstanding the high prevalence of CMD, effective treatment 128 remains elusive. Therapeutic interventions such as nicorandil, statins, and angiotensin- 129 converting enzyme inhibitors may offer potential benefits [3, 4]. However, the complete 130 clinical picture remains incompletely understood. Traditional Chinese medicine (TCM), 131 which has been utilized in clinical practice throughout China for nearly two millennia, has 132 shown promise as a clinically viable approach for treating CMD [5, 6]. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 7 / 58 133 The development of CMD is significantly influenced by structural and/or functional 134 abnormalities in cardiac microvascular endothelial cells (CMECs) [7]. In a physiological 135 setting, the coronary microcirculation serves as the primary resistance artery in the 136 coronary arteries. And it plays a crucial role in regulating coronary blood flow (CBF) [8]. 137 CMECs, which constitute approximately one-third of all heart cells [9], are essential 138 components of the coronary microcirculation [7], They play a critical role in controlling CBF 139 and maintaining proper coronary microvascular function [10, 11]. When stimulated by 140 pathological factors, CMECs lose their ability to proliferate, adhere, migrate normally or 141 undergo apoptosis and secrete substances as usual. This can lead to abnormal contraction 142 and diastolic function in microvessels as well as compromised integrity of the 143 microvascular barrier and thinning of coronary microarterioles [12, 13]. Ultimately, this may 144 result in reduced coronary flow reserve (CFR) and inadequate myocardial blood supply 145 [14]. Therefore, investigating the mechanism through which compound Chinese medicine 146 (CCM) and its constituents intervene in CMECs under various injury models can provide 147 valuable insights into the potential of TCM for treating CMD. This review aims to present 148 the latest evidence on TCM intervention mechanisms in CMECs under diverse injury 149 models. 150 2. Methods 151 The systematic literature review was conducted in accordance with the guidelines 152 outlined by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 153 (PRISMA) statement [15] (S1 Table). The systematic review has been registered in the 154 INPLASY platform for prospective registration with the registration number .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 8 / 58 155 INPLASY202470092. Protocol details are available at INPLASY Protocol 6561 – INPLASY. 156 Ethical approval was not required due to no human subjects being involved in this study. 157 2.1. Search Strategy 158 From database construction until March 2024, the electronic databases utilized for 159 literature search encompassed PubMed, Embase, Web of Science, Scopus, China 160 National Knowledge Infrastructure and China Biology Medicine disc. The keywords 161 employed were “Chinese herbal medicine monomer”, “Chinese herbal medicine 162 components”, “Chinese herbal compound”, “traditional Chinese medicine”, “herbal 163 medicine”, “Chinese herbal medicine”, “Chinese herb”, “CHM”, “TCM”, “China Chinese 164 herbal medicine”, “China extract”, “China fraction”, “China formula”, “China prescription, 165 "CMECs", "CMEC", "coronary microvascular endothelial cells", "cardiac microvascular 166 endothelial cells", "myocardiac microvascular endothelial cells". The search scope was 167 limited to full-text articles without any additional restrictions. 168 2.2. Flowchart Sketch of the Screening Process 169 The flowchart illustrating the systematic review screening process is presented in Fig 170 1. Initially, a total of 296 literature sources were identified and evaluated. Following a de- 171 weighting procedure, 84 papers were excluded, while 212 papers met the inclusion criteria. 172 The inclusion criteria consisted of the following: 1) the literature had to be written in English; 173 2) it had to be the full text; 3) the content of the literature had to be related to Chinese 174 herbal medicines (CHM); 4) the study design had to involve CMECs or cardiac 175 microvasculature. On the other hand, the exclusion criteria encompassed irrelevant 176 literature, reviews, meta-analyses, case reports, conference proceedings, book chapters, .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 9 / 58 177 letters to the editor, oral presentations, posters, and editorials. During the initial screening, 178 128 papers were excluded based on a skim of the title or abstract, leaving 84 papers for 179 further evaluation. Among these, 12 papers lacked full text in English, 7 papers did not 180 involve CMECs or cardiac microvessels in their design, and 2 papers were not focused on 181 heart-related research. These 21 papers were consequently excluded. Finally, a total of 63 182 relevant studies were included in this review. Among them, 19 studies employed models 183 of ischemic or hypoxic injury, 16 studies utilized models of ischemia/reperfusion (I/R) or 184 hypoxia/reoxygenation (H/R) injury, 10 studies employed models of inflammatory injury, 185 and 18 studies employed models of other types of injury, based on cellular processing 186 methods (S2 Table). This review aims to comprehensively report and critically analyze the 187 relevant studies. However, it should be noted that a meta-analysis was not conducted. 188 Fig 1. PRISMA flow chart. 189 3. Results 190 Among the 64 papers included, the intervention methods employed for CMECs 191 exhibited considerable variation. Based on the specific cellular treatments applied, the 192 literature can be categorized into distinct injury models, including ischemic or hypoxic injury, 193 I/R or H/R injury, inflammatory injury, metabolic injury, angiotensin II (Ang II) injury, as well 194 as other treatments. In these studies, a total of 16 CCMs and 18 major bioactive 195 components (MBCs) were included. The CCMs and MBCs that are effective in regulating 196 CMECs under various injury models are listed in S3 and S4 Tables. In the subsequent 197 paragraphs, we will delve into a comprehensive review of the roles played by CCM and its 198 constituents in different injury models, along with the underlying pathway mechanisms .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 10 / 58 199 implicated. 200 3.1. TCM that Modify CMECs in Ischemic or Hypoxic 201 Injury Model 202 Ischemia- and hypoxia-induced injury is widely regarded as the underlying 203 pathological basis and initial process of numerous cardiovascular conditions. Protecting 204 CMECs from injury caused by ischemia and hypoxia represents a crucial therapeutic 205 approach for addressing a range of cardiovascular diseases [16]. Studies have discovered 206 that TCM compounds or core constituents in regulating this injury plays a certain influence. 207 and the involved mechanisms are illustrated in Fig 2 and summarized in Table 1. 208 Fig 2. TCM regulate various signaling pathways that mediate CMECs dysfunction 209 induced by hypoxia or ischemia. Akt, Protein kinase B; ALDH, Aldehyde dehydrogenase; 210 ATP, Adenosine triphosphate; bFGF, Basic fibroblast growth factor; CCM, Compound 211 Chinese medicine; CL, Carthamus tinctorius L. and Lepidium apetalum Willd; DGBX, 212 DangGuiBuXue Tang; EGb761, Ginkgo biloba extract; eNOS, Endothelial nitric oxide 213 synthase; ERK, Extracellular signal-regulated kinase; DG, GeGen DanShen extract; HIF, 214 Hypoxia-inducible factor; IRF5, Interferon regulatory factor 5; KLF, Krüppel-like factor; 215 MDG-1, A water-soluble beta-D-fructan from O. japonicus; MI, Myocardial infarction; 216 mTOR, Mammalian target of rapamycin; NRG-1, Neuregulin-1; OGD, Oxygen glucose 217 deprivation; PI3K, Phosphoinositide 3-kinase; QLQX, QiLiQiangXin; QSYQ, QiShenYiQi; 218 S1P, Sphingosine 1 phosphate; STDP, Shexiang Tongxin Dropping Pill; SXBX, 219 SheXiangBaoXin Pill; Syk, Spleen tyrosine kinase; TGF, Transforming growth factor; TJs, 220 Tight junctions; TXL, TongXinLuo; VA, Velvet Antler; VAP, Velvet Antler Proteins; VEGF, .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 11 / 58 221 Vascular endothelial growth factor; VEGFR, Vascular endothelial growth factor receptor. 222 Table 1. Effects of CCM, CMM and MBC injury induced by hypoxia or ischemic. CCM, CMM and MBC In vitro/In vivo Model (cells) Model (animals) Signaling pathways Effects References CL Both Rat CMECs (Hypoxia 48 hours) SD male rats (Left anterior descending coronary ligation- induced MI） TGFβ1/Snail signaling pathway 8;9; Zhou et al. (2023) QLQX In vitro Rat CMECs (Hypoxia 24 hours) / miR-21/HIF- 1α/VEGF signaling pathway 10; Wang et al. (2021) In vitro Rat CMECs (Hypoxia 12 hours) / HIF-1α/VEGF signaling pathway; AMPK/mTOR/HIF-1α signaling pathway 1;2;3; 10; Wang et al. (2018) In vitro Rat CMECs (Hypoxia 12 hours) / NRG-1/ErbB signaling pathway; PI3K/AKT/mTOR signaling pathway; HIF-1α/VEGF signaling pathway 3;10; Wang J. F. et al. (2017) QSYQ In vitro Rat CMECs was from myocardial infarction in rat which using the ligature method / mir-223- 3p/RPS6KB1/HIF-1α signaling pathway 10; Dai et al. (2016) STDP In vivo / SD male rats (Left anterior descending coronary ligation- induced MI） Dectin-1/Syk/IRF5 signaling pathway 4;6;11; Cui et al. (2023) DGBX In vivo / SD male rats (Left anterior descending coronary ligation- induced MI） VEGF/VEGFRs signaling pathway 10; Hu et al. (2018) .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 12 / 58 TXL Both Human CMECs (Hypoxia 3 days) SD male rats (Left anterior descending coronary ligation- induced MF) NRG-1/ErbB signaling pathway; PI3K/AKT signaling pathway 8; Yin et al. (2019) In vitro Human CMECs (stimulated with CoCl2 for 24 hours) / H3K9ac/claudin-9 6; Liu et al. (2016) / KLF4, TJ proteins 6; Zheng et al. (2015)In vitro Human CMECs (stimulated with CoCl2 for 24 hours) In vitro Human CMECs (stimulated with CoCl2) / HIF-2α/VEGF signaling pathway 2;4; Li Y. N. et al. (2015) DG In vivo / SD male rats (Left anterior descending coronary ligation- induced MI) VEGF/VEGFR2 signaling pathway 10; Zhai et al. (2021) EGb 761 In vivo / 16.5-17 months male wistar rats (Hypoxia 20min) / 15; Welt et al. (1996) VA In vivo / SD male rats (Left anterior descending coronary ligation- induced MI） Notch signaling pathway; VEGF signaling pathway 10; Li et al. (2018) VAP In vitro Rat CMECs (Hypoxic-Ischemic 46 hours) / PI3K/AKT signaling pathway 3;10; Xiao et al. (2017) SXBX In vitro Rat CMECs (Hypoxia 24 hours) / ALDH2/AKT/mTOR signaling pathway 3;10; Hu et al. (2021) MDG-1 In vitro Human CMECs (OGD 8 hours) / S1P/bFGF/AKT/ERK /eNOS signaling pathway 10; Wang S. O. et al. (2010) Both Human CMECs (OGD 8 hours) SD male rats (Paraconal interventricular branch of the left coronary ligation） S1P/AKT/ERK signaling pathway 9; Wang et al. (2012) 223 Akt, Protein kinase B; ALDH, Aldehyde dehydrogenase; bFGF, Basic fibroblast .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 13 / 58 224 growth factor; CCM, Compound Chinese medicine; CL, Carthamus tinctorius L. and 225 Lepidium apetalum Willd; CMECs, Cardiac microvascular endothelial cells; CMM, 226 Chinese materia medica; DGBX, DangGuiBuXue Tang; EGb 761, Ginkgo biloba 227 extract; eNOS, Endothelial nitric oxide synthase; ERK, Extracellular signal-regulated 228 kinase; DG, GeGen DanShen extract; HIF, Hypoxia-inducible factor; IRF5, Interferon 229 regulatory factor 5; KLF, Krüppel-like factor; MBC, Major bioactive component; MDG- 230 1, A water-soluble beta-D-fructan from O. japonicus; MI, Myocardial infarction; 231 mTOR, Mammalian target of rapamycin; NRG-1, Neuregulin-1; OGD, Oxygen 232 glucose deprivation; PI3K, Phosphoinositide 3-kinase; QLQX, QiLiQiangXin; QSYQ, 233 QiShenYiQi; S1P, Sphingosine 1 phosphate; SD, Sprague-Dawley; STDP, 234 SheXiangTongXin Dropping Pill; SXBX, SheXiangBaoXin Pill; Syk, Spleen tyrosine 235 kinase; TGF, Transforming growth factor; TJs, Tight junctions; TXL, TongXinLuo; VA, 236 Velvet Antler; VAP, Velvet Antler Proteins; VEGF, Vascular endothelial growth factor; 237 VEGFR, Vascular endothelial growth factor receptor; 1, Regulation of energy 238 metabolism; 2, Attenuation of oxidative stress; 3, Anti-apoptosis; 4, Inhibition of 239 inflammatory cytokines release; 5, Regulation of adhesion molecule expression; 6, 240 Alleviation microvascular hyperpermeability; 7, Repression autophagy; 8, Inhibition of 241 fibrosis; 9, Controlling both paracrine and autocrine processes; 10, Promotion of 242 microvascular generation; 11, Dilation of coronary microvessels; 12, Cell senescence 243 mitigation; 13, Reducing potential microthrombosis; 14, Anti-pyroptosis; 15, 244 Improving the structure of the cell. 245 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 14 / 58 246 Ischemia or hypoxia can potentially impede angiogenesis. Animal studies have shown 247 that Velvet Antler (VA), GeGen DanShen extract (DG), and DangGuiBuXue Tang (DGBX) 248 can effectively mitigate the damage caused by myocardial infarction (MI) and enhance 249 microvessel density within the infarct zone [17-19]. SheXiangTongXin Dropping Pill (STDP) 250 demonstrated the ability to prevent microvascular leakage, reduce edema, hemorrhage, 251 and inflammatory cell infiltration in the tissues surrounding the microvessels, while 252 preserving the morphological integrity of myocardial microvessels [20]. Another study 253 discovered that Ginkgo biloba extract (EGb761) can protect the ultrastructure of CMECs 254 against hypoxia [21]. 255 Hypoxia-inducible factor (HIF) is recognized as the major transcriptional regulator of 256 the adaptive response to hypoxia [22]. The presence of HIF-1α stimulates the expression 257 of various angiogenic factors, including transforming growth factor (TGF)-α, platelet- 258 derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) [23]. 259 QiLiQiangXin (QLQX) has been shown to protect CMECs from hypoxia-induced injury by 260 promoting angiogenesis and CMECs proliferation through the activation of the miR-21/HIF- 261 1α/VEGF axis and HIF-1α-mediated glycolysis promotion [24]. In an alternative study, 262 QLQX was found to induce the adenosine 5'-monophosphate-activated protein kinase 263 (AMPK)/mammalian target of rapamycin (mTOR)/HIF-1α signaling pathway, leading to 264 increased HIF-1α protein expression in hypoxic CMECs. The stability of HIF-1α and its 265 activation of genes are closely associated with prolyl hydroxylase domain enzymes (PHDs) 266 [25], and QLQX was found to downregulate the expression of PHDs, thereby enhancing 267 HIF-1α stability [26]. Neuregulin-1 (NRG-1), a cardioactive growth factor released from .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 15 / 58 268 endothelial cells, is indispensable for cardiac development, structural maintenance, and 269 functional integrity of the heart. They transmit their signals through interactions with cell 270 membrane receptors of the ErbB family [27]. QLQX activates the phosphatidylinositol 3- 271 kinase (PI3K)/protein kinase B (AKT)/mTOR signaling pathway, which further activates the 272 HIF-1α/VEGF signaling pathway to protect against hypoxia. This mechanism is mediated 273 by the NRG-1/ErbB signaling pathway [28]. 274 TongXinLuo (TXL) has been previously investigated as a potential treatment for luo 275 illness. Both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are 276 inducible enzymes involved in oxidative stress and inflammation [29, 30]. In hypoxia- 277 induced human CMECs, TXL was found to inhibit the production of VEGF, HIF-2α, COX- 278 2, and iNOS. High doses of TXL were found to inhibit hypoxia-induced increases in the 279 levels of the inflammatory mediator prostaglandin E2 (PGE2) and the oxidative marker 280 nitrotyrosine (NT), thereby attenuating inflammation and oxidative damage. However, TXL 281 did not enhance the expression of prostacyclin synthase (PGIS) or endothelial nitric oxide 282 synthase (eNOS) [31]. PGIS possesses anti-inflammatory properties and cytoprotective 283 effects [32], while eNOS expression promotes blood vessel dilation [33]. Macrophages 284 have been implicated in hypoxia-induced injury to human CMECs, and peroxynitrite is 285 involved in this process. Another study found that TXL can increase the expression of PGIS 286 primarily and reduce endothelin-converting enzyme-1 (ECE-1) expression through 287 inhibiting macrophage-mediated nitrotyrosine accumulation [34]. Notably, ECE-1 plays a 288 crucial role in the signaling pathway for endothelin and serves as a significant modulator 289 of vascular tone [35]. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 16 / 58 290 The integrity of the microvascular barrier relies on various factors, including the 291 subendothelial basement membrane, caveolin quantity and function in endothelial cells, 292 and the presence of adherens junctions (AJs) and tight junctions (TJs) between adjacent 293 CMECs [36]. TJs proteins encompass occludin, claudin, and junctional adhesion molecule 294 (JAM), which are transmembrane proteins [37] AJs are responsible for calcium-mediated 295 homophilic adhesion between neighboring cells through cadherin, including zona 296 occludens 1 (ZO-1) protein, β-catenin, vascular endothelial (VE)-cadherin, among others 297 [38, 39]. TXL can reverse the hypoxia-inhibited claudin-9 expression by elevating histone 298 H3 lysine 9 acetylation (H3K9ac) in the promoter region of its gene [40]. Furthermore, TXL 299 provides protection by enhancing the phosphorylation of krüppel-like factor (KLF) 4, a basic 300 transcription factor that regulates the production of occludin, claudin-1, VE- cadherin, and 301 β-catenin [41]. 302 The transcriptional repressor snail can induce the endothelial-to-mesenchymal 303 transition (EndMT). The resultant EndMT-derived cardiac fibroblasts (CFs), synthesize 304 large amounts of collagen, which contributes to fibrotic lesions and abnormal collagen fiber 305 deposition [42]. TXL inhibits EndMT by downregulating snail expression in CMECs after 306 three days of hypoxia and activates the NRG-1/ErbB/PI3K/AKT signaling pathway to 307 reduce myocardial fibrosis (MF) following acute MI [43]. Hypoxia for 48 hours activates the 308 TGF-β1/snail signaling pathway, and the Carthamus tinctorius L. and Lepidium apetalum 309 Willd drug pair (CL) can inhibit EndMT in CMECs by inhibiting the TGF-β1/snail signal 310 pathway [44]. 311 QiShenYiQi (QSYQ) downregulate the expression of mir-223-3p, activate the .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 17 / 58 312 ribosomal protein S6 kinase B1 (RPS6KB1)/HIF-1α signaling pathway, and facilitate 313 ischemic cardiac angiogenesis [45]. A study on Ophiopogon japonicus and its water- 314 soluble beta-D-fructan (MDG-1) revealed that the sphingosine 1 phosphate 315 (S1P)/AKT/extracellular signal-regulated kinase (ERK) signaling pathway might be 316 involved in the potential mechanism of their anti-ischemic action on cell survival [46]. Basic 317 fibroblast growth factor (bFGF) is a prominent factor that stimulates endothelial cell 318 proliferation and neovascularization by binding to the fibroblast growth factor (FGF) 319 receptor [47]. Another investigation demonstrated that MDG-1 could promote angiogenesis 320 by protecting human CMECs and cardiomyocytes from ischemia-induced cellular damage 321 through the S1P/bFGF/AKT/ERK/eNOS signaling pathway [48]. SheXiangBaoXin Pill 322 (SXBX) promotes angiogenesis through the aldehyde dehydrogenase 323 (ALDH)2/AKT/mTOR signaling pathway [49]. Velvet antler proteins (VAP) safeguards 324 CMECs against ischemia-hypoxia by modulating the PI3K/AKT signaling pathway [50]. 325 3.2. TCM that Modify CMECs in I/R or H/R Injury Model 326 I/R-induced damage to the microcirculation can lead to increased leukocyte adhesion, 327 reactive oxygen species (ROS) production, dysfunction of CMECs, and leakage of 328 macromolecules. These events can trigger inflammatory responses, impair CBF, and 329 ultimately result in cardiomyocyte death. Therefore, the preservation of CMECs and the 330 reduction of CMD following I/R are considered promising approaches in the treatment of 331 cardiac I/R injury [51]. 332 Studies have demonstrated that TCM can inhibit apoptosis, regulate the expression 333 of adhesion molecules, alleviate microvascular permeability, dilate coronary microvessels, .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 18 / 58 334 mitigate microvascular obstruction, modulate oxidative stress, and control inflammation, 335 among other effects. According to research on animals, the DanLou formula (DLF) and the 336 TongMaiYangXin pill (TMYX) enhance myocardial no-reflow following I/R [52, 53]. 337 Resveratrol (RSV) and scutellarin (SCU) have been found to regulate the production of 338 intracellular proteins and the paracrine secretion function of CMECs following H/R damage 339 [54, 55]. The specific effects and underlying mechanisms of action are illustrated in Fig 3 340 and summarized in Table 2. 341 Fig 3. TCM regulate various signaling pathways that mediate CMECs dysfunction 342 induced by I/R or H/R. AKT, Protein kinase B; AP, Astragalus polysaccharide; ASIV, 343 Astragaloside IV; ATM, Ataxia telangiectasia mutated; ATP, Adenosine triphosphate; Drp1, 344 Dynamin-related protein 1; EGb761, Ginkgo biloba extract; eNOS, Endothelial nitric oxide 345 synthase; ERK, Extracellular signal-regulated kinase; GAS, Gastrodin; H/R, 346 Hypoxia/Reoxygenation; I/R, Ischemia/reperfusion; IGF1R, Insulin-like growth factor 1 347 receptor; iNOS, Inducible nitric oxide synthase; IRS1, Insulin receptor substrate 1; MAPK, 348 Mitogen-activated protein kinase; MEK, Mitogen-activated protein kinase; Mff, 349 Mitochondrial fission factor; NLRP3, Nucleotide-binding oligomerization domain-like 350 receptor protein 3; NR4A1, Nuclear receptor subfamily 4 group A member 1; PI3K, 351 Phosphoinositide 3-kinase; PINK, Phosphatase and tensin homolog-induced putative 352 kinase; QSYQ, QiShenYiQi; SCU, Scutellarin; SL, ShenLian extract; SSNX, 353 ShuangShenNingXin formula; TXL, TongXinLuo. 354 Table 2. Effects of CCM, CMM and MBC injury induced by I/R or H/R. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 19 / 58 CCM, CMM and MBC In vitro/in vivo Model (cells) Model (animals) Signaling pathways Effects Reference s SL Both Rat CMECs （OGD 2 hours and reoxygenation and resupply of glucose 2 hours） Wistar male rats （Ischemia 2 hours and reperfusion 24 hours） PINK/Parkin signaling pathway 4;6;7;13; Li et al. (2023) ASIV Both Human CMECs （Hypoxia 2 hours and reoxygenation 2 hours） SD male rats （Ischemia 30min and reperfusion 90 min） IGF1R/IRS1/PI3K/A KT signaling pathway 1;6; He et al. (2022) AP In vitro Human CMECs （OGD 2 hours and reoxygenation and resupply of glucose 24 hours） / p38 MAPK signaling pathway 5; Zhu et al. (2013) RSV In vitro Human CMECs （Hypoxia 12 hours and reoxygenation 2 hours） / / 3;4;9; Cui et al. (2022) QSYQ Both Human CMECs （Hypoxia 2 hours and reoxygenation 3 hours） SD male rats （Ischemia 30min and reperfusion 90 min） Src/caveolin-1 signaling pathway; RhoA/ROCK/MLC signaling pathway 1;6;9; Pan et al. (2021) SSNX Both CMECs （OGD 4 hours and reoxygenation and resupply of glucose 2 hours） SD male rats （Ischemia 45 min and reperfusion 24 hours） NR4A1/mff/Drp1 signaling pathway 1;3;10;1 1; Liu et al. (2024) .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 20 / 58 GAS Both Human CMECs （Hypoxia 2 hours and reoxygenation 2 hours） I/R NLRP3/caspase-1 signaling pathway 4;14; Sun et al. (2019) EGb 761 In vitro Rat CMECs （Hypoxia 2 hours and reoxygenation） / ATM signaling pathway 2;3; Zhang et al. (2017) TXL Both Human CMECs （Hypoxia 18 hours and reoxygenation 2 hours） SD male rats （Ischemia 45 min and reperfusion 3 hours） miR-145- 5p/p70s6k1/eNOS signaling pathway 11; Chen GH. et al. (2020) In vitro Human CMECs （Hypoxia 2 hours and reoxygenation 2 hours） / / 3; Li Q. et al. (2017) In vitro Human CMECs （Hypoxia 12 hours and reoxygenation 2 hours） / / 3;9; Cui et al. (2016) In vitro Human CMECs （Hypoxia 2 hours and reoxygenation 2 hours） / MEK/ERK signaling pathway 3;7; Cui et al. (2014) SCU In vitro Human CMECs （Hypoxia 12 hours and reoxygenation 12 hours） / / 9; Shi et al. (2015) .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 21 / 58 DLF in vivo / SD male rats （Ischemia 60 min and reperfusion 2 hours） eNOS/iNOS signaling pathway 11;13; Dai et al. (2021) TMYX in vivo / SD male rats （Ischemia 2 hours and reperfusion） PI3K/AKT/eNOS signaling pathway 11; Chen R. et al. (2020) extracts from 12 Chinese edible flowers In vitro Rat CMECs （Hypoxia 1 hours and reoxygenation 1 hours） / / 2;3; Wang F. et al. (2017) 355 AKT, Protein kinase B; AP, Astragalus polysaccharide; ASIV, Astragaloside IV; ATM, 356 Ataxia telangiectasia mutated; CCM, Compound Chinese medicine; CMECs, Cardiac 357 microvascular endothelial cells; CMM, Chinese materia medica; DLF, DanLou formula; 358 Drp1, Dynamin-related protein 1; EGb761, Ginkgo biloba extract; eNOS, Endothelial nitric 359 oxide synthase; ERK, Extracellular signal-regulated kinase; GAS, Gastrodin; H/R, 360 Hypoxia/Reoxygenation; I/R, Ischemia/reperfusion; IGF1R, Insulin-like growth factor 1 361 receptor; iNOS, Inducible nitric oxide synthase; IRS1, Insulin receptor substrate 1; MAPK, 362 Mitogen-activated protein kinase; MBC, Major bioactive component; MEK, Mitogen- 363 activated protein kinase; Mff, Mitochondrial fission factor; NLRP3, Nucleotide-binding 364 oligomerization domain-like receptor protein 3; NR4A1, Nuclear receptor subfamily 4 group 365 A member 1; OGD, Oxygen glucose deprivation; PI3K, Phosphoinositide 3-kinase; PINK, 366 Phosphatase and tensin homolog-induced putative kinase; QSYQ, QiShenYiQi; RSV, .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 22 / 58 367 Resveratrol; SCU, Scutellarin; SL, ShenLian extract; SSNX, ShuangShenNingXin formula; 368 TMYX, TongMaiYangXin pill; TXL, TongXinLuo; 1, Regulation of energy metabolism; 2, 369 Attenuation of oxidative stress; 3, Anti-apoptosis; 4, Inhibition of inflammatory cytokines 370 release; 5, Regulation of adhesion molecule expression; 6, Alleviation microvascular 371 hyperpermeability; 7, Repression autophagy; 8, Inhibition of fibrosis; 9, Controlling both 372 paracrine and autocrine processes; 10, Promotion of microvascular generation; 11, Dilation 373 of coronary microvessels; 12, Cell senescence mitigation; 13, Reducing potential 374 microthrombosis; 14, Anti-pyroptosis; 15, Improving the structure of the cell. 375 376 Through the ras homolog gene family member A (RhoA)/Rho-associated protein 377 kinase (ROCK)/myosin light chain (MLC) signaling pathway, QSYQ have been found to 378 inhibit I/R-induced cardiac microvascular hyperpermeability. Src family protein tyrosine 379 kinases are known to be associated with endothelial cell permeability [56]. QSYQ has been 380 shown to attenuate the upregulation of Src, p-caveolin-1, matrix metallopeptidase-9 (MMP- 381 9), and cathepsin S (CTSS) induced by H/R, while also preserving the expression of 382 claudin-5. Among the signaling pathways involved in mediating the effects of QSYQ, the 383 Src/caveolin-1 pathway has been implicated [57]. 384 Astragaloside IV (ASIV), the main active ingredient in QSYQ, has been found to 385 protect the microvascular endothelial barrier. ASIV mitigates adenosine triphosphate (ATP) 386 depletion, enhances the expression of tight junction proteins between endothelial cells, 387 and promotes the H/R-induced activation of the insulin-like growth factor 1 receptor (IGF1R) 388 and downstream phosphorylation of insulin receptor substrate 1 (IRS1)/PI3K/AKT .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 23 / 58 389 signaling pathway [58]. 390 Following I/R, the release of cytokines, oxygen radicals, and pro-inflammatory 391 mediators stimulates the vascular endothelium and neutrophils. This leads to the 392 upregulation of adhesion molecules, promoting the adhesion and migration of white blood 393 cells across the vascular endothelium [59]. Astragalus polysaccharide (AP), a key 394 component of QSYQ, reduces the expression of relevant adhesion molecules following I/R. 395 AP also inhibits the interaction between human CMECs and polymorphonuclear leukocyte 396 (PMN) during I/R by suppressing the p38 mitogen-activated protein kinase (MAPK) 397 signaling pathway and downregulating the expression of adhesion molecules (P-selectin 398 and E-selectin) in human CMECs [60]. 399 TXL has been shown to exert a protective effect against H/R injury, inhibit apoptosis 400 in CMECs, and regulate protein expression and paracrine function in these cells [61, 62]. 401 In cardiomyocytes treated with TXL following H/R, vesicles containing long intergenic non- 402 coding RNA regulator of reprogramming (Linc-ROR) are released and taken up by CMECs. 403 Subsequently, Linc-ROR downregulates its target miR-145-5p, which in turn promotes the 404 production of 70 kDa ribosomal protein S6 kinase 1 (P70S6k1) and activates the eNOS 405 pathway in CMECs [63]. Autophagy is an essential cellular process involved in the 406 degradation of aging or dysfunctional organelles and protein aggregates, serving as a 407 quality control mechanism. Impaired autophagy leads to the accumulation of dysfunctional 408 organelles and proteins, resulting in endoplasmic reticulum stress (ERS) and apoptosis 409 [64]. TXL has been found to induce autophagy via activation of the mitogen-activated 410 protein kinase (MEK)/ERK signaling pathway, thereby protecting human CMECs from H/R .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 24 / 58 411 injury [65]. 412 I/R injury in CMECs induces signaling pathways involved in both mitochondrial division 413 and apoptosis. ShenLian extract (SL) has been shown to prevent mitochondrial autophagy 414 and preserve mitochondrial activity, thereby reducing endothelial cell death, preserving 415 endothelial cell function, and protecting the microvasculature, which ultimately helps 416 mitigate coronary artery no-reflow. The phosphatase and tensin homolog-induced putative 417 kinase (PINK)/Parkin signaling pathway is implicated in this process [66]. 418 ShuangShenNingXin formula (SSNX) restores mitochondrial division to normal levels 419 and inhibits mitochondrial apoptosis, thereby reducing potential damage to the 420 mitochondrial membrane and preventing its opening. The mechanism underlying the 421 effects of SSNX may involve the nuclear receptor subfamily 4 group A member 1 422 (NR4A1)/mitochondrial fission factor (Mff)/dynamin-related protein 1 (Drp1) signaling 423 pathway [67]. NR4A1 has been shown to regulate mitochondrial fission [68]. Mff is known 424 to stimulate mitochondrial fission by recruiting Drp1, a critical protein involved in the control 425 of mitochondrial division. Pathological mitochondrial fission in CMECs can trigger 426 unfavorable mitochondrial apoptotic pathways [69]. 427 Oxidative stress is a crucial factor in I/R injury, and ROS play a central role in 428 mediating oxidative stress [70]. EGb761 has been shown to inhibit the I/R-induced 429 activation of the ataxia telangiectasia mutated (ATM) pathway, thereby ameliorating 430 apoptosis by suppressing ROS expression [71]. In a study evaluating the antioxidant 431 effects of 12 edible flowers. Most of the edible flowers examined demonstrated the 432 potential to enhance the antioxidant capacity of CMECs following I/R. Honeysuckle, rose, .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 25 / 58 433 and wild chrysanthemum were found to exhibit the highest levels of antioxidant activity 434 [72]. 435 The nucleotide-binding oligomerization domain-like receptor protein 3 436 (NLRP3)/caspase-1 signaling pathway is believed to play a critical role in the injury to 437 CMECs and cardiac tissues, as well as the increase in infarct area during I/R injury. 438 Gastrodin (GAS) has been shown to partially reverse the pyroptosis of CMECs and reduce 439 the area of MI and inflammatory cell infiltration by inhibiting the NLRP3/caspase-1 signaling 440 pathway [73]. 441 3.3. TCM that Modify CMECs in Inflammatory Injury 442 Model 443 Vascular inflammation plays a significant role in the pathogenesis of CMD [74], It can 444 lead to damage in coronary microvessels, as well as promote thrombosis and perivascular 445 fibrosis. The vascular endothelium, acting as a barrier between the vascular lumen and 446 surrounding tissue, serves as a key regulator and participant in the vascular inflammatory 447 response [75]. Inflammatory substances such as tumor necrosis factor-α (TNF-α), 448 homocysteine (Hcy), and lipopolysaccharide (LPS) are known to induce inflammation in 449 cells [76-78]. TCM exerts its effects on enhancing CMECs following vascular inflammatory 450 injury mainly through modulation of the nuclear factor-kappa B (NF-κB), MAPKs, and Janus 451 tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) signaling 452 pathways [79, 80]. For instance, ShenMai formula (SMF), 4-O-(2-O-acetyl-6-O-p- 453 coumaroyl-β-D-glucopyranosyl)-p-coumaric acid (4-ACGC) isolated from Bidens pilosa 454 Linn., and salidroside (SA) have been shown to impact the development of vascular .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 26 / 58 455 inflammation [76, 81, 82]. Fig 4 and Table 3 provide a visual representation of the 456 underlying mechanisms and the expression of relevant inflammatory mediators. 457 Fig 4. TCM regulate various signaling pathways that mediate CMECs dysfunction 458 induced by inflammation. 4-ACGC, 4-O-(2″-O-acetyl-6″-O- p-coumaroyl-β-D- 459 glucopyranosyl) -p-coumaric acid; AND, Andrographolide; ASIV, Astragaloside IV; CYP, 460 Alpha-Cyperone; CYT, Caffeoylxanthiazonoside; Hcy, Homocysteine; HO-1, Heme 461 oxygenase-1; IL, Interleukin; JAK, Janus tyrosine kinase; KLF, Krüppel-like factor; LPS, 462 lipopolysaccharide; MAPK, Mitogen-activated protein kinase; MDA, Malondialdehyde; NF- 463 κB, Nuclear factor-kappa B; Nrf2, Nuclear factor-erythroid 2 related factor 2; QLQX, 464 QiLiQiangXin; SA, Salidroside; SD, Sprague Dawley; SMF, ShenMai formula; SOD, 465 Superoxide Dismutase; STAT, Signal transduction and activator of transcription; TNF-α, 466 Tumor necrosis factor-α; TNF-α, Tumor necrosis factor-α; TXD, TianXiangDan; TXL, 467 TongXinLuo; XQT, XiangQiTang. 468 Table 3. Effects of CCM, CMM and MBC injury induced by inflammatory. CCM, CMM and MBC In vitro/in vivo Model (cells) Model (animals) Signaling pathways Effects Inflammatory mediators References TXD Both Human CMECs (LPS) SD rats（Inject sodium laurate was injected into the left ventricle） Nrf2/HO-1 signaling pathway; NF-κB signaling pathway 4;10; ↓IL-1β, ↓TNF-α Sawuer et al. (2021) QLQX In vitro Human CMECs (Hcy) / JAK/STAT signaling pathway 4;10; ↓IL-6 Zhang et al. (2019) TXL In vitro Human CMECs (C16) / NF-κB signaling pathway 2;4; ↓IL-1β, ↓TNF-α, ↓MDA, ↓SOD Wu et al. (2015) .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 27 / 58 SA In vitro Rat CMECs (TNF-α) / MAPK signaling pathway; NF-κB signaingl pathway 4;5; ↓VCAM-1, ↓IL- 1β, ↓IL-6, ↓MCP- 1 Li et al. (2019) 4-ACGC Both Rat CMECs (TNF-α) SD male rats (Left anterior descending coronary ligation） / 4; ↓IL-1β, ↓IL-6 Yang et al. (2018) SMF In vitro Rat CMECs (LPS) / NF-κB signaling pathway 4;5; ↓IL-1, ↓IL-6, ↓TNF-α, ↓ICAM- 1, ↓LDH Zhu et al. (2017) CYT Both Rat CMECs (LPS) SD male rats (Left anterior descending coronary ligation） NF-κB signaling pathway 4; ↓TNF-α, ↓IL-1β, ↓IL-6 Yang et al. (2017) AND In vitro Rat CMECs (LPS) / / 1;4; ↓IL-6, ↓TNF-α Feng et al. (2017) In vitro Rat CMECs (LPS) / MAPK signaling pathway; KLF2mRNA/NF- κB signaling pathway 4;5;13; ↓TNF-α, ↓ICAM- 1, ↓PAI-1 He et al. (2013) CYP In vitro Rat CMECs (LPS) / MAPK signaling pathway; KLF2mRNA/NF- κB signaling pathway 4;5;13; ↓TNF-α, ↓ICAM- 1, ↓PAI-1 He et al. (2013) XQT In vitro Rat CMECs (LPS) / MAPK signaling pathway; KLF2mRNA/NF- κB signaling pathway 4;5;13; ↓TNF-α, ↓ICAM- 1, ↓PAI-1 He et al. (2013) ASIV In vitro Rat CMECs (LPS) / MAPK signaling pathway; KLF2mRNA/NF- kB signaling pathway 4;5;13; ↓TNF-α, ↓ICAM- 1, ↓PAI-1 He et al. (2013) .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 28 / 58 BTE In vitro Human CMECs (IL-1β) / / 4; ↓IL-6 Lakota et al. (2009) RSV In vitro Human CMECs (IL-1β) / / 4; ↓IL-6, ↓prostacyclin Lakota et al. (2009) 469 4-ACGC, 4-O-(2″-O-acetyl-6″-O- p-coumaroyl-β-D-glucopyranosyl) -p-coumaric acid; AND, 470 Andrographolide; ASIV, Astragaloside IV; BTE, Black tea extract; CCM, Compound 471 Chinese medicine; CMECs, Cardiac microvascular endothelial cells; CMM, Chinese 472 materia medica; CYP, Alpha-Cyperone; CYT, Caffeoylxanthiazonoside; Hcy, 473 Homocysteine; HO-1, Heme oxygenase-1; ICAM, Intercellular cell adhesion molecule; IL, 474 Interleukin; JAK, Janus tyrosine kinase; KLF, Krüppel-like factor; LDH, Lactate 475 dehydrogenase; LPS, lipopolysaccharide; MAPK, Mitogen-activated protein kinase; MBC, 476 Major bioactive component; MDA, Malondialdehyde; NF-κB, Nuclear factor-kappa B; Nrf2, 477 Nuclear factor‑erythroid 2 related factor 2; PAI, Plasminogen activator inhibitor; QLQX, 478 QiLiQiangXin; RSV, Resveratrol; SA, Salidroside; SD, Sprague Dawley; SMF, ShenMai 479 formula; SOD, Superoxide Dismutase; STAT, Signal transduction and activator of 480 transcription; TNF-α, Tumor necrosis factor α; TXD, TianXiangDan; TXL, TongXinLuo; 481 XQT, XiangQiTang; 1, Regulation of energy metabolism; 2, Attenuation of oxidative stress; 482 3, Anti-apoptosis; 4, Inhibition of inflammatory cytokines release; 5, Regulation of adhesion 483 molecule expression; 6, Alleviation microvascular hyperpermeability; 7, Repression 484 autophagy; 8, Inhibition of fibrosis; 9, Controlling both paracrine and autocrine processes; 485 10, Promotion of microvascular generation; 11, Dilation of coronary microvessels; 12, Cell 486 senescence mitigation; 13, Reducing potential microthrombosis; 14, Anti-pyroptosis; 15, 487 Improving the structure of the cell. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 29 / 58 488 489 The inhibitory effect of TianXiangDan (TXD) on LPS-induced microvascular 490 endothelial inflammation has been associated with the activation of nuclear factor-erythroid 491 2-related factor 2 (Nrf2). Heme oxygenase-1 (HO-1) is one of the downstream proteins in 492 the Nrf2 signaling pathway. TXD exerts its anti-inflammatory effects by suppressing the 493 expression of TNF-α, phosphorylated inhibitor of kappa B alpha (p-IκBα), phosphorylated 494 p65 (p-p65), and interleukin (IL)-1β through the induction of HO-1 protein [83]. 495 C16 induction in cells leads to oxidative stress and inflammation. Among the various 496 sources of intracellular ROS, Nicotinamide adenine dinucleotide phosphate (NADPH) 497 oxidase plays a significant role [84, 85]. TXL inhibits the release of TNF-α and IL-1β 498 induced by C16 through the blockade of NF-κB activation and expression. Moreover, TXL 499 suppresses the upregulation of malondialdehyde (MDA) and superoxide dismutase (SOD), 500 thereby reducing the production of ROS. Its antioxidant activities may be attributed to the 501 suppression of HO-1 and NADPH oxidase complex expression in human CMECs [86]. 502 Different levels of NF-κB p65 expression and regulation are observed in the cytoplasm 503 and nucleus. When exposed to LPS, the fruit of Xanthium strumarium L plant contains an 504 active ingredient called caffeoylxanthiazonoside (CYT), which significantly reduces the 505 production of TNF-α, IL-1β, and IL-6. Additionally, there is an upregulation of inhibitor of 506 NF-κB (IκB）and cytoplasmic NF-κB p65 protein expression, along with a downregulation 507 of nuclear NF-κB p65 [87]. 508 Inflammation and coagulation are closely related and can mutually promote each other 509 [88]. Plasminogen activator inhibitor (PAI)-1 [89] and tissue factor (TF) [90] are important .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 30 / 58 510 factors in the coagulation cascade. XiangQiTang (XQT) and its components, Alpha- 511 Cyperone (CYP), ASIV, and andrographolide (AND), inhibit LPS-stimulated production of 512 TNF-α, intercellular adhesion molecule-1, and PAI-1. They also upregulate KLF2 mRNA 513 expression, reduce the phosphorylation level of NF-κB p65 protein, and inhibit TF secretion. 514 Moreover, XQT, CYP, ASIV, and AND suppress the expression of proteins involved in the 515 MAPK signaling pathway [91]. 516 Caveolin-1 is a crucial structural protein that facilitates the transmembrane transport 517 of low-density lipoprotein cholesterol (LDL-C), thereby promoting the development of 518 atherosclerosis [92]. AND significantly reduces caveolin-1 expression in LPS-induced 519 CMECs. It modulates the expression of IL-6 and TNF-α among inflammatory factors and 520 inhibits extracellular ATP-induced calcium release by decreasing the expression of 521 phospholipase Cδ3 (PLCδ3), without affecting extracellular calcium endocytosis. This has 522 a limited impact on nitric oxide (NO) production and release [93]. 523 Lakota et al.[94] demonstrated that human CMECs responded dose-dependently to 524 IL-1β-induced IL-6 levels and prostacyclin release. The administration of black tea extract 525 (BTE) and RSV inhibited IL-1β-induced responses. Additionally, QLQX downregulated the 526 expression of phosphorylated STAT3, phosphorylated JAK2, and IL-6 in CMECs. It 527 upregulated the expression of vascular-endothelial growth factor A (VEGFA), mitigated the 528 inflammatory process induced by Hcy, and promoted angiogenesis, potentially through 529 modulation of the JAK/STAT signaling pathway [95]. 530 3.4. TCM that Modify CMECs in Metabolic Injury Model 531 Cellular metabolism is essential for maintaining normal cellular biochemical processes .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 31 / 58 532 and biological activities. Patients with metabolic abnormalities, such as elevated levels of 533 free fatty acids and chronic hyperglycemia, face an augmented risk of cardiovascular 534 events [96]. 535 A high fat diet (HFD) is a significant risk factor for organ damage, including the liver, 536 kidneys, and heart, leading to increased mortality [97]. HFD can induce capillary 537 permeability in the microvascular environment, potentially leading to interstitial fibrosis and 538 myocardial dysfunction [98]. In mice fed a HFD, chronic intermittent administration of 539 quercetin (Q) reduces intramyocardial fat accumulation, increases cardiac microvessel 540 density, and regulates oxidative stress [99]. 541 Apolipoprotein E (apoE) deficiency is known to cause elevated levels of cholesterol- 542 rich compounds in the bloodstream, contributing to the development of atherosclerotic 543 lesions [100]. Treatment with TXL significantly decreases lipid levels, reduces 544 atherosclerotic plaque formation in apoE-deficient mice, and improves endothelial cell 545 function [86]. 546 In animal models, N(ω)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase 547 (NOS) inhibitor, suppresses NOS activity and reduces NO production, leading to increased 548 blood pressure and the progression of left ventricular remodeling [101]. QSYQ has been 549 found to increase myocardial capillary density and inhibit microvascular endothelial 550 inflammation induced by L-NAME combined with the HFD [102]. 551 A high glucose (HG) environment can downregulate the expression of claudins-5 and 552 -11 in human CMECs. TXL can reverse the HG-induced inhibition of claudins-5 and -11 by 553 increasing H3K9ac in the promoters of these genes. Moreover, high-dose TXL treatment .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 32 / 58 554 promotes the membrane localization of claudins-5 and -11 in HG-stimulated human 555 CMECs [103]. 556 Angiopoietin-like 4 (Angptl4) plays a protective role in regulating the endothelial barrier, 557 maintaining vascular integrity by preserving the VE-calmodulin complex [104]. In the 558 context of preserving the structure and function of the endothelial barrier under conditions 559 of high glucose-induced I/R, TXL has been found to be comparable to insulin and 560 recombinant human Angptl4. The expression of Angptl4 can be induced by peroxisome 561 proliferator-activated receptor α (PPAR-α). In diabetic patients, TXL may preserve the 562 integrity of the endothelial barrier against I/R injury through the activation of the PPAR- 563 α/Angptl4 signaling pathway [105, 106]. 564 3.5. TCM that Modify CMECs in Ang II Injury Model 565 Ang II is a bioactive peptide that regulates vascular tone and promotes the proliferation 566 of vascular smooth muscle cells, playing a pivotal role in the pathogenesis of 567 cardiovascular disease. Ang II has been implicated in inducing cardiac hypertrophy [107], 568 a which is an independent risk factor for mortality [108]. Studies have found that Ang II- 569 induced apoptosis in CMECs is closely associated with the development of CMD in heart 570 failure patients [109]. It has been observed that Ang II enhances endothelial cell apoptosis, 571 impairs the shear response of CMECs, and hampers the morphological adaptation to shear 572 stress by downregulating the expression of platelet-endothelial cell adhesion molecule-1 573 (PECAM-1). Allicin (A), through stimulation of the PECAM-1/PI3K/AKT/eNOS signaling 574 pathway, downregulation of caspase-3 and receptor interacting protein 3 (RIP3) 575 expression, and prevention of necrotic apoptosis, enhances the functionality of CMECs. A .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 33 / 58 576 was found to increase microvessel density in rats with cardiac hypertrophy induced by 577 abdominal aortic constriction [110]. 578 Autophagy is a cyclic process involved in maintaining cellular homeostasis [111]. The 579 Forkhead-Box Class O (FoxO) family member, FoxO3a, regulates autophagy by activating 580 genes involved in autophagosome formation [112]. CMECs exposed to Ang II undergo 581 apoptosis, but QLQX prevents this by inhibiting autophagy through the ErbB2/AKT/FoxO3a 582 signaling pathway [113]. In terms of the microvascular endothelial barrier, TXL attenuates 583 the damage to human CMECs induced by Ang II by promoting KLF5 expression, which 584 enhances the levels of tight junction proteins [114]. 585 3.6. TCM that Modify CMECs in Other Injury Models 586 Aging is considered an independent factor associated with endothelial cell dysfunction 587 [115]. TCM has shown promise in alleviating senescence in CMECs. From a cytoskeletal 588 perspective, age affects the structure and function of F-actin. Extracts of Panax 589 Notoginseng (PN), Radix Ginseng (RG), and Rhizoma Ligustici Chuanxiong (RLC) have 590 been found to delay the senescence of CMECs in response to heat shock protein 27 591 (HSP27) and reduce F-actin synthesis [116]. 592 Viral myocarditis (VMC) is a common cardiovascular disease [117]. Chronic phase 593 VMC is characterized by MF [118]. CFs are the most affected cells in MF [119]. It has been 594 discovered that CFs can also arise from the EndMT process, which may contribute to MF 595 development in VMC [120]. Ginsenoside-Rb3 (Rb3), a major component of Sanqi and 596 Renshen, inhibits EndMT in CMECs after coxsackievirus B3 (CVB3) infection through the 597 proline-rich tyrosine kinase (Pyk) 2/PI3K/AKT signaling pathway [121]. Ginsenoside-Rg3 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 34 / 58 598 (Rg3) activates AKT to upregulate the Nrf2/antioxidant response element (ARE) pathway, 599 thereby mitigating cardiotoxicity caused by adriamycin (ADM) and ameliorating endothelial 600 dysfunction resulting from oxidative stress [122]. Millettia pulchra Kurz var.laxior (Dunn) Z. 601 Wei is a wild plant from the Fabaceae family with diverse therapeutic uses. Its root contains 602 the flavonoid monomer 17-Methoxyl-7-hydroxy-benzene-furanchalcone (MHBFC) [123]. In 603 an animal model, MHBFC attenuated L-NAME-induced apoptosis of CMECs during 604 cardiac remodeling in rats [124]. Diosmetin-7-O-β-D-glucopyranoside (Diosmetin-7-O- 605 glucoside) is a natural flavonoid abundant in citrus fruits and herbal extracts like fructus 606 trichosanthes peel [125]. In primary CMECs, TGF-β1 promoted EndMT. Diosmetin-7-O- 607 glucoside, partly through an Src-dependent mechanism, regulates EndMT via endoplasmic 608 reticulum stress [126]. 609 Some studies have focused on the direct intervention of Chinese medicine or active 610 ingredients without specifically addressing CMEC injury. For instance, the chemical 611 constituents of Ophiopogon japonicus (OJ) fiber root and DG were found to modulate 612 angiogenesis in human CMECs, promoting microvessel formation [127, 128]. In terms of 613 regulating microvascular function, long-term oral treatment with Oroxylin A (OA), the 614 primary constituent of Radix Scutellariae (RS), was found to enhance the production of NO 615 and the expression of eNOS protein in CMECs, as well as the production of NO and the 616 expression of iNOS protein in vascular smooth muscle cells (VSMCs). It was proposed that 617 the mechanism of action of OA involved the modulation of the estrogen receptor (ER) 618 signaling pathway [129]. Tanshinone Ⅱ A (Tan Ⅱ A) was found to activate the ER 619 signaling pathway in primary CMECs, leading to increased expression of eNOS gene, NO .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 35 / 58 620 production, ERK1/2 phosphorylation, and Ca2+ mobilization [130]. In the treatment of heart 621 failure (HF), periplocin (PER) in Cortex Periplocae Sepii Radicis was compared with the 622 cardiac glycoside ouabain. PER was found to increase cell proliferation, reduce cell 623 damage, inhibit apoptosis, and affect the expression of guanosine triphosphate (GTP)- 624 binding proteins, which are closely related to intracellular calcium signaling. The underlying 625 mechanism may involve the protein serine/threonine kinase pathway, cellular metabolism, 626 and other cellular processes [131]. 627 4. Outlook for Future Research 628 CMD poses a significant challenge to achieving clinical benefits in IHD, and effective 629 interventions are currently limited, providing an opportunity for intervention with TCM. In 630 recent years, several studies have demonstrated that TCM can improve CMD by protecting 631 CMECs against various injuries. These interventions have shown structural improvements 632 such as increased microvessel density and number, reduced microthrombosis, and 633 inhibition of endothelial-to-mesenchymal transition. Functionally, TCM has been found to 634 dilate coronary microvessels, alleviate microvascular permeability, and delay cellular 635 senescence in CMECs. The mechanisms of action involve antioxidant, anti-apoptotic, anti- 636 inflammatory effects, as well as regulation of energy metabolism, among others. 637 Considering that disease manifestation is often the result of multiple cell types within 638 the same tissue or involving other tissues, co-culture systems have been used to mimic 639 this condition, where different cell types share the same culture environment [132]. Recent 640 studies have highlighted the ability of TCM to modulate the paracrine and autocrine 641 secretion of CMECs, thereby influencing their own behavior and that of surrounding cells. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 36 / 58 642 Enhancing the crosstalk between different cell types represents an important area for 643 future research. Additionally, it has been observed that women have a higher risk of CMD 644 compared to men [133], emphasizing the importance of studying gender differences in the 645 pathophysiology of CMD [134]. Currently, preclinical studies rarely consider gender as a 646 variable in their analyses [135]. Therefore, investigating the effects of CMD on CMECs in 647 different genders may present a new research direction. 648 5. Limitations 649 The present study has several limitations that should be acknowledged. Firstly, the 650 specific chemical structures of the active ingredients present in the Chinese medicines 651 discussed in some articles remain unknown. Secondly, there are variations in the 652 intervention methods used for compound Chinese medicines or the core components of 653 Chinese medicines across different studies, leading to variations in the observed effects. 654 Therefore, it is still necessary to establish standardized and uniform intervention methods 655 to ensure consistency and comparability among studies. 656 6. Summary 657 This systematic review provides evidence that different injury models lead to distinct 658 phenotypes in CMECs, and the intervention mechanisms of TCM also vary accordingly. 659 Specifically, under ischemic or hypoxic injury conditions, TCM demonstrates its efficacy by 660 promoting microangiogenesis, alleviating microvascular permeability, and inhibiting 661 myocardial fibrosis. These effects are mediated through signaling pathways such as HIF- 662 1α/VEGF, PI3K/AKT, and Snail. In the context of H/R injury, TCM exerts its benefits by 663 inhibiting apoptosis, alleviating microvascular permeability, and dilating coronary .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint 37 / 58 664 microvessels, involving pathways such as ATM, PI3K/AKT, p70s6kT, p70s6kT/eNOS, and 665 others. In the case of inflammatory injury, TCM acts by suppressing the release of 666 inflammatory cytokines, regulating the expression of adhesion molecules, and reducing the 667 formation of microthrombosis, through pathways such as MAPK, NF-κB, and JAK/STAT. 668 Furthermore, under metabolic injury, angiotensin II, aging, and other pathological 669 conditions, TCM demonstrates its efficacy by alleviating microvascular permeability, 670 dilating coronary microvessels, and inhibiting inflammation and oxidative stress. The 671 underlying signaling pathways involved include PPAR-α/Angptl4, H3K9ac/claudins, 672 PI3K/AKT, among others. This systematic review provides a comprehensive overview of 673 the effects of TCM on CMECs in various injury models and highlights the associated 674 signaling pathway studies. 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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 8, 2024. ; https://doi.org/10.1101/2024.09.05.611361doi: bioRxiv preprint