Single-cell analysis reveals synergistic therapeutic mechanisms of Shenqi Fuzheng formula against acute myocardial infarction

i

This article has been updated. A Correction to this article was published on .

i

The Original Article was published on

i

The Original Article was published on

i

This article has been updated

i

This article was retracted on

i

This article has been updated

Abstract

The Shenqi Fuzheng (SQ) formula, made from Codonopsis Radix (Dangshen, DS) and Astragali Radix (Huangqi, HQ), has been used in Traditional Chinese Medicine (TCM) and exhibits cardio-protective effects against acute myocardial infarction (AMI). However, the molecular basis of DS-HQ synergy remains unclear. Here, we combined RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) to explore their synergistic mechanisms. Transcriptomic analyses showed that SQ influenced the AMI network more effectively than either herb alone. ScRNA-seq analysis revealed that these synergistic effects involve both direct and indirect pro-angiogenic pathways. The combination treatment enhanced pro-angiogenic responses in microvascular endothelial subpopulations and promoted angiogenesis in the border zone. It also enhanced the anti-inflammatory role of a specific reparative macrophage subset and stimulated vascular endothelial growth factor A (VEGFA) secretion from these cells, supporting the VEGFA-VEGFR signaling between macrophages and microvascular endothelial cells. This coordinated regulation provides a plausible mechanistic explanation for the reduced infarct size and improved cardiac outcomes seenin vivo, highlighting the therapeutic value of TCM.

Keywords

Shenqi Fuzheng formula / Efficacy enhancement / Synergistic mechanism / Acute myocardial infarction / Chemical composition / Single-cell RNA sequencing

CHANGE HISTORY

A Correction to this paper has been published:
https://doi.org

The Retraction Note was updated to reflect reception of statements of disagreement from all authors.

This article has been retracted.Please see the Retraction Notice for more detail:
https://doi.org

References

[1] Wang ML, Zhu BJ, Gao M, et al. Identification of chemical constituents and blood-absorbed components of Shenqi Fuzheng extract based on UPLC-Triple-TOF/MS technology. Future Integr Med. 2024; 3:222-238. https://doi.org/10.14218/fim.2024.00037.

[2] Liao J, Hao C, Huang WH, et al. Network pharmacology study reveals energy metabolism and apoptosis pathways-mediated cardioprotective effects of Shenqi Fuzheng. J Ethnopharmacol. 2018; 227:155-165. https://doi.org/10.1016/j.jep.2018.08.029.

[3] Du J, Cheng BCY, Fu X, et al. In vitro assays suggest Shenqi Fuzheng Injection has the potential to alter melanoma immune microenvironment. J Ethnopharmacol. 2016; 194:15-19. https://doi.org/10.1016/j.jep.2016.08.038.

[4] Ma YR, Bai BF, Liu D, et al. Shenqi Fuzheng Injection reduces cisplatin-induced kidney injury via cGAS/STING signaling pathway in breast cancer mice model. Breast Cancer (Dove Med Press). 2024; 16:451-469. https://doi.org/10.2147/bctt.S475860.

[5] Guo W, Liu S, Xia H, et al. Shenqi Fuzheng injection facilitates skeletal muscle mitophagy mediated by the ubiquitination of HIF-1α to ameliorate cancer-associated fatigue. J Cell Mol Med. 2024; 28:e18455. https://doi.org/10.1111/jcmm.18455.

[6] Xiao D, Zhang HZ, Zuo XH, et al. Observation on the efficacy of Shenqi Fuzheng Injection in treating senile heart failure after acute myocardial infarction. Chin J Integr Med Cardio Cerebrovsc Dis. 2016; 14:1061-1063. https://doi.org/10.3969/j.issn.1672-1349.2016.10.002.

[7] Yun ML. Clinic observation of 55 cases with chronic heart failure treated by shenqi fuzheng injection. Nat Sci J Hainan Univ. 2006; 24:371-373. https://doi.org/1004-1729(2006)04-0371-03.

[8] liu P, Xu XY, Li M . Effect of shenqi fuzheng injection on peripheral blood ANGII, ET-1 levels and clinical curative effect in patients with congestive heart failure. Chin J Biochem Pharm. 2015; 35:81-83. https://doi.org/1005-1678(2015)12-0081-03.

[9] Wang L, Ye BH, Wang DX, et al. Effects of Shenqi Fuzheng Injection on plasma N terminal pro-brain natriuretic peptide in patients with chronic heart failure. Pract J Cardiac Cereb Pneum Vasc Dis. 2011; 19:1774-1775. https://doi.org/1008-5971(2011)10-1774-02.

[10] Tsao CW, Aday AW, Almarzooq ZI, et al. Heart disease and stroke statistics-2023 update: a report from the American heart association. Circulation. 2023; 147:e622. https://doi.org/10.1161/cir.0000000000001123.

[11] Zhou MG, Wang HD, Zeng XY, et al. Mortality, morbidity, and risk factors in China and its provinces, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2019; 394:1145-1158. https://doi.org/10.1016/s0140-6736(19)30427-1.

[12] Wang XW, Guo ZK, Ding ZF, et al. Inflammation, autophagy, and apoptosis after myocardial infarction. J Am Heart Assoc. 2018; 7:e008024. https://doi.org/10.1161/jaha.117.008024.

[13] Ruiz-Villalba A, Romero JP, Hernández SC, et al. Single-cell RNA sequencing analysis reveals a crucial role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) cardiac fibroblasts after myocardial infarction. Circulation. 2020; 142:1831-1847. https://doi.org/10.1161/circulationaha.119.044557.

[14] Yu Q, Guo DW, Peng JN, et al. Prevalence and adverse outcomes of frailty in older patients with acute myocardial infarction after percutaneous coronary interventions: a systematic review and meta-analysis. Clin Cardiol. 2022; 46:5-12. https://doi.org/10.1002/clc.23929.

[15] Takura T, Komuro I, Ono M . Trends in the cost-effectiveness level of percutaneous coronary intervention: macro socioeconomic analysis and health technology assessment. J Cardiol. 2023; 81:356-363. https://doi.org/10.1016/j.jjcc.2022.09.011.

[16] Al Said S, Kaier K, Sumaya W, et al. Non-vitamin-K-antagonist oral anticoagulants (NOACs) after acute myocardial infarction: a network meta-analysis. Cochrane Database Syst Rev. 2024; 1:e014678. https://doi.org/10.1002/14651858.CD014678.pub2.

[17] Jiang BP, Si JY, Luo HM, et al. Single-cell omics reveal the mechanisms of traditional Chinese medicines. Phytomedicine. 2025; 147:157204. https://doi.org/10.1016/j.phymed.2025.157204.

[18] Gu YC, Wang GJ, Pan GY, et al. Transport and bioavailability studies of astragaloside IV, an active ingredient in Radix Astragali. Basic Clin Pharmacol Toxicol. 2004; 95:295-298. https://doi.org/10.1111/j.1742-7843.2004.t01-1-pto950508.x.

[19] Du Y, Zhang Q, Chen G, et al. Pharmacokinetics of Astragaloside IV in rats by liquid chromatography coupled with tandem mass spectrometry. Eur J Drug Metab Pharmacokinet. 2005; 30:269-273. https://doi.org/10.1007/BF03190631.

[20] Matsui J, Yamamoto Y, Funahashi Y, et al. E7080, a novel inhibitor that targets multiple kinases, has potent antitumor activities against stem cell factor producing human small cell lung cancer H146, based on angiogenesis inhibition. Int J Cancer. 2008; 122:664-671. https://doi.org/10.1002/ijc.23131.

[21] Foucquier J, Guedj M . Analysis of drug combinations: current methodological landscape. Pharmacol Res Perspect. 2015; 7:e00549. https://doi.org/10.1002/prp2.149.

[22] Vlot AHC, Aniceto N, Menden MP, et al. Applying synergy metrics to combination screening data: agreements, disagreements and pitfalls. Drug Discov Today. 2019; 24:2286-2298. https://doi.org/10.1016/j.drudis.2019.09.002.

[23] Wu LH, Li X, Yang JH, et al. CHD@ZJU: a knowledgebase providing network-based research platform on coronary heart disease. Database (Oxford). 2013; 2013:bat047. https://doi.org/10.1093/database/bat047.

[24] Wu LH, Wang Y, Li Z, et al. Identifying roles of Jun-Chen-Zuo-Shi component herbs of QiShenYiQi formula in treating acute myocardial ischemia by network pharmacology. Chin Med. 2014; 9:24. https://doi.org/10.1186/1749-8546-9-24.

[25] Xu XJ, Long JB, Jin KY, et al. Danshen-Chuanxiongqin Injection attenuates cerebral ischemic stroke by inhibiting neuroinflammation via the TLR2/TLR4-MyD88-NF-κB Pathway in tMCAO mice. Chin J Nat Med. 2021; 19:772-783. https://doi.org/10.1016/s1875-5364(21)60083-3.

[26] Hao Y, Stuart T, Kowalski MH, et al. Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat Biotechnol. 2023; 42:293-304. https://doi.org/10.1038/s41587-023-01767-y.

[27] Han C, Shi CP, Liu LM, et al. Majorbio Cloud 2024: update single-cell and multiomics workflows. iMeta. 2024; 3:e217. https://doi.org/10.1002/imt2.217.

[28] Jin SQ, Guerrero-Juarez CF, Zhang LH, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 2021; 12:1088. https://doi.org/10.1038/s41467-021-21246-9.

[29] Hathaway CK, Grant R, Hagaman JR, et al. Endothelin-1 critically influences cardiac function via superoxide-MMP9 cascade. Proc Natl Acad Sci USA. 2015; 112:5141-5146. https://doi.org/10.1073/pnas.1504557112.

[30] Clancy RM, Markham AJ, Jackson T, et al. Cardiac fibroblast transcriptome analyses support a role for interferogenic, profibrotic, and inflammatory genes in anti-SSA/Ro-associated congenital heart block. Am J Physiol Heart Circ Physiol. 2017; 313:631-640. https://doi.org/10.1152/ajpheart.00256.2017.

[31] Zheng M, Liu ZY, Liu NN, et al. The effect of Wenxin Keli on the mRNA expression profile of rabbits with myocardial infarction. Evid Based Compl Alt. 2016; 2016:2352614. https://doi.org/10.1155/2016/2352614.

[32] Wang YH, Xian HM . Identifying genes related to acute myocardial infarction based on network control capability. Genes (Basel). 2022; 13:1238. https://doi.org/10.3390/genes13071238.

[33] Li S, Tao B, Wan JJ, et al. A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction. Cell Rep Med. 2024; 5:101795. https://doi.org/10.1016/j.xcrm.2024.101795.

[34] Yang SY, Pei LY, Huang ZJ, et al. Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction. Mol Med. 2024; 30:89. https://doi.org/10.1186/s10020-024-00856-1.

[35] Wu XK, Reboll MR, Korf-Klingebiel M, et al. Angiogenesis after acute myocardial infarction. Cardiovasc Res. 2021; 117:1257-1273. https://doi.org/10.1093/cvr/cvaa287.

[36] Gladka MM, Molenaar B, de Ruiter H, et al. Single-cell sequencing of the healthy and diseased heart reveals cytoskeleton-associated protein 4 as a new modulator of fibroblasts activation. Circulation. 2018; 138:166-180. https://doi.org/10.1161/CIRCULATIONAHA.117.030742.

[37] Kim JO, Park JH, Kim T, et al. A novel system-level approach using RNA-sequencing data identifies miR-30-5p and miR-142a-5p as key regulators of apoptosis in myocardial infarction. Sci Rep. 2018; 8:14638. https://doi.org/10.1038/s41598-018-33020-x.

[38] Grover SP, Mackman N . Tissue factor in atherosclerosis and atherothrombosis. Atherosclerosis. 2020; 307:80-86. https://doi.org/10.1016/j.atherosclerosis.2020.06.003.

[39] Mao XL, Xu H, Liu XL, et al. Proteomics of urinary exosomes for discovering novel non-invasive biomarkers of acute myocardial infarction patients. Int J Biol Macromol. 2025; 302:140427. https://doi.org/10.1016/j.ijbiomac.2025.140427.

[40] Budas GR, Churchill EN, Disatnik MH, et al. Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury. Cardiovasc Res. 2010; 88:83-92. https://doi.org/10.1093/cvr/cvq154.

[41] Ke D, Cao MZ, Ni J, et al. Macrophage and fibroblast trajectory inference and crosstalk analysis during myocardial infarction using integrated single-cell transcriptomic datasets. J Transl Med. 2024; 22:560. https://doi.org/10.1186/s12967-024-05353-x.

[42] Farbehi N, Patrick R, Dorison A, et al. Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury. eLife. 2019; 8:e43882. https://doi.org/10.7554/eLife.43882.

[43] Tallquist MD . Cardiac fibroblast diversity. Annu Rev Physiol. 2020; 82:63-78. https://doi.org/10.1146/annurev-physiol-021119-034527.

[44] Yap J, Irei J, Lozano-Gerona J, et al. Macrophages in cardiac remodelling after myocardial infarction. Nat Rev Cardiol. 2023; 20:373-385. https://doi.org/10.1038/s41569-022-00823-5.

[45] Jung SH, Hwang BH, Shin S, et al. Spatiotemporal dynamics of macrophage heterogeneity and a potential function of Trem2^hi macrophages in infarcted hearts. Nat Commun. 2022; 13:4580. https://doi.org/10.1038/s41467-022-32284-2.

[46] Jin KY, Gao S, Yang PH, et al. Single-cell RNA sequencing reveals the temporal diversity and dynamics of cardiac immunity after myocardial infarction. Small Methods. 2022; 6:e2100752. https://doi.org/10.1002/smtd.202100752.

[47] Tani H, Sadahiro T, Yamada Y, et al. Direct reprogramming improves cardiac function and reverses fibrosis in chronic myocardial infarction. Circulation. 2023; 147:223-238. https://doi.org/10.1161/CIRCULATIONAHA.121.058655.

[48] Ivey MJ, Kuwabara JT, Pai JT, et al. Resident fibroblast expansion during cardiac growth and remodeling. J Mol Cell Cardiol. 2018; 114:161-174. https://doi.org/10.1016/j.yjmcc.2017.11.012.

[49] Straub AC, Lohman AW, Billaud M, et al. Endothelial cell expression of haemoglobin alpha regulates nitric oxide signalling. Nature. 2012; 491:473-477. https://doi.org/10.1038/nature11626.

[50] Ninh VK, Calcagno DM, Yu JD, et al. Spatially clustered type I interferon responses at injury borderzones. Nature. 2024; 633:174-181. https://doi.org/10.1038/s41586-024-07806-1.

[51] Tombor LS, John D, Glaser SF, et al. Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction. Nat Commun. 2021; 12:681. https://doi.org/10.1038/s41467-021-20905-1.

[52] Martin P, Leibovich SJ . Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol. 2005; 15:599-607. https://doi.org/10.1016/j.tcb.2005.09.002.

[53] Epelman S, Lavine KJ, Beaudin AE, et al. Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation. Immunity. 2014; 40:91-104. https://doi.org/10.1016/j.immuni.2013.11.019.

[54] Bajpai G, Bredemeyer A, Li WJ, et al. Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial injury. Circ Res. 2019; 124:263-278. https://doi.org/10.1161/CIRCRESAHA.118.314028.

[55] Das A, Sinha M, Datta S, et al. Monocyte and macrophage plasticity in tissue repair and regeneration. Am J Pathol. 2015; 185:2596-2606. https://doi.org/10.1016/j.ajpath.2015.06.001.

[56] Ferraro B, Leoni G, Hinkel R, et al. Pro-angiogenic macrophage phenotype to promote myocardial repair. J Am Coll Cardiol. 2019; 73:2990-3002. https://doi.org/10.1016/j.jacc.2019.03.503.

[57] Lu XY, Yao JK, Li CX, et al. Shexiang Tongxin Dropping Pills promote macrophage polarization-induced angiogenesis against coronary microvascular dysfunctionvia PI3K/Akt/mTORC1 pathway. Front Pharmacol. 2022; 13:840521. https://doi.org/10.3389/fphar.2022.840521.

[58] Tallquist MD, Molkentin JD . Redefining the identity of cardiac fibroblasts. Nat Rev Cardiol. 2017; 14:484-491. https://doi.org/10.1038/nrcardio.2017.57.

[59] Shinde AV, Frangogiannis NG . Mechanisms of fibroblast activation in the remodeling myocardium. Curr Pathobiol Rep. 2017; 5:145-152. https://doi.org/10.1007/s40139-017-0132-z.

[60] Fu X, Khalil H, Kanisicak O, et al. Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart. J Clin Invest. 2018; 128:2127-2143. https://doi.org/10.1172/JCI98215.

[61] Sun Y, Weber KT . Infarct scar: a dynamic tissue. Cardiovasc Res. 2000; 46:250-256. https://doi.org/10.1016/s0008-6363(00)00032-8.

[62] Yin YF, Tan MY, Han LH, et al. The hippo kinases MST1/2 in cardiovascular and metabolic diseases: a promising therapeutic target option for pharmacotherapy. Acta Pharm Sin B . 2023; 13:1956-1975. https://doi.org/10.1016/j.apsb.2023.01.015.

[63] Cheng SY, Zhang XX, Feng Q, et al. Astragaloside IV exerts angiogenesis and cardioprotection after myocardial infarction via regulating PTEN/PI3K/Akt signaling pathway. Life Sci. 2019; 227:82-93. https://doi.org/10.1016/j.lfs.2019.04.040.

[64] Pan L, Zhang XF, Wei WS, et al. The cardiovascular protective effect and mechanism of calycosin and its derivatives. Chin J Nat Med. 2020; 18:907-915. https://doi.org/10.1016/S1875-5364(20)60034-6.

[65] Wang JN, Kan CD, Lee LT, et al. Herbal extract from Codonopsis pilosula (Franch.) Nannf. enhances cardiogenic differentiation and improves the function of infarcted rat hearts. Life (Basel). 2021; 11:422. https://doi.org/10.3390/life11050422.

[66] Chen XY, Shao R, Wang Y . Multi-layered effects of Codonopsis Radix on the immune system. Acupunct Herb Med. 2024; 4:405-419. https://doi.org/10.1097/hm9.0000000000000130.