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Sodium Fluoride Exposure Induces Developmental Toxicity and Cardiotoxicity in Zebrafish Embryos.
| Biological Trace Element Research | Wang F, Chen F, Song W, Li Y, Wu H, Tian T, Tian M, Tang D, Liu Y. |
Developmental toxicity RNA Zebrafish Heart Immune System Animal Study
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Abstract

Full-text study online at
https://link.springer.com/article/10.1007/s12011-024-04381-4

Fluorosis is a worldwide public health problem, in which the heart is an important target organ. However, studies on its toxicological mechanism in embryonic development are limited. This study assessed the toxicity of sodium fluoride (NaF) toward zebrafish embryos. We determined the mortality, hatching rate, phenotypic malformation, heart function, and morphology of zebrafish embryos after exposure to NaF. Subsequently, the molecular mechanism was revealed using high-throughput RNA sequencing analysis. The expression levels of key genes for heart development were detected using quantitative real-time reverse transcription PCR. The 50% lethal concentration (LC50) value of NaF toward zebrafish embryos at 96 h post-fertilization was 335.75 mg/L. When the concentration of NaF was higher than 200 mg/L, severe deformities, such as pericardial edema, yolk sac edema, spine curvature, shortened body length, reduced head area, and eye area, were observed. The heart rate of the embryos exposed to NaF decreased in a dose-dependent fashion. The distance between the sinus venosus and bulbus arteriosus was significantly increased in the NaF-exposed group compared with that in the control group. The stroke volume and cardiac output decreased significantly in the NaF groups. Compared with the control group, the expression levels of Gata4, Tbx5a, Hand2, Tnnt2c, Nppa, and Myh6 were significantly increased in the NaF-treated group. Through transcriptome sequencing, 1354 differentially expressed genes (DEGs) were detected in the NaF (200 mg/L) treated groups, including 1253 upregulated genes and 101 downregulated genes. Gene ontology functional analysis and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the DEGs showed that cardiac-related pathways, such as actin cytoskeleton regulation, Jak-Stat, PI3k-Akt, and Ras, were activated in the NaF-exposed group. This study revealed the underlying mechanism of fluoride-induced cardiac morphological and functional abnormalities and provides clues for the clinical prevention and treatment of fluorosis.

EXCERPTS:

Zebrafish (Danio rerio), a small tropical freshwater teleost, is an ideal model organism to study vertebrate congenital heart disease [14]. Its features of short generation time, high fecundity, rapid development, and transparency during early developmental make it easy to carry out rapid toxicity assessment in vivo [15, 16]. In addition, zebrafish have similar cardiac developmental processes, cardiac function, and heart disease characteristics to humans [17]. It has been reported that homologs of about 70% of human genes can be found in zebrafish, and more than 80% of proteins that cause human diseases have homologs in zebrafish [18]. However, there has been no report on the effect of fluorine on zebrafish cardiac function and its regulatory mechanism.

In this study, we explored the cardiac teratogenicity and developmental toxicity of NaF using the zebrafish embryo model. The mechanism of NaF toxicity in the zebrafish embryonic heart was analyzed by transcriptomic analysis. This study revealed the mechanism of the cardiotoxicity caused by fluorosis and provided research data for the prevention and treatment of cardiac injury caused by fluorosis.

Toxic Effects of Fluorosis on Juvenile Zebrafish

The body length, head area, eye area, and pericardium area of each group of larvae were measured at 120 hpf to evaluate the developmental toxicity of NaF toward larvae. The obvious morphological changes were shortening of the body length (Fig. 3A), decreased head area (Fig. 3B) and eye area (Fig. 3C), and increased pericardium area (Fig. 3D). All of these phenotypes were dose-dependent.

Differential Gene Expression Caused by Sodium Fluoride

RNA-seq analysis was used to unravel the underlying mechanisms of NaF toxicity in zebrafish. After sequencing, we first evaluated the quality of sequencing data using FastQC, followed by Trimmomatic analysis to obtain clean data (Table S2). HISAT2 was used to compare the clean data of the samples with the reference genome and count the mapping information (Table S3). We found that 1116 (4.02%) genes were specifically expressed and 709 (2.55%) genes were absent in the NaF exposure group compared with the control group (Fig. 6A). Zebrafish differentially expressed genes (DEGs) were selected using DEGseq, with the filter conditions of qValue?<?0.05 and |log2FoldChange|>?1. Compared with the control group, the NaF (200 mg/L) exposure group had 1253 upregulated genes and 101 downregulated genes (Fig. 6B). A heatmap of significant DEGs upon NaF treatment is shown in Fig. 6C.

Discussion

Epidemiological survey results show that compared with those in residents in low-fluoride areas, the rates of heart disease and mortality of residents in high-fluoride areas are significantly higher [13]. However, the molecular mechanism of fluoride-induced heart disease is unknown. We utilized the zebrafish larvae as a model to assess the toxic effects of NaF on cardiac development. Our findings suggest that NaF exposure causes cardiac developmental malformations, including pericardial edema, and cardiac structural and functional impairment.

The heart is the first organ to form and plays a critical role during the development of embryos. The zebrafish heart is mature and functional at 72 hpf [22,23,24], and its morphological structure and function are similar to the human heart during the early stage of development [25]. In recent years, zebrafish have been widely used as a vertebrate model to research human cardiovascular disease [26]. In addition, they are widely used for environmental toxicity detection and drug toxicity safety evaluation [27, 28]. The heart must undergo a series of complex processes as it matures, and any defect in these processes can lead to congenital heart disease [29]. The evaluation of NaF o is necessary for safety and zebrafish is a well-accepted animal model for researching “predictive toxicology” [30]. Early embryos are often used to assess toxicity during embryonic development [31]. Embryos (6 hpf) were distributed into six-well plates and incubated with NaF at the designated concentrations in this study. Zebrafish exposed to NaF exhibited severe heart malformation, such as pericardial edema, cardiac loop deformity, and an increased SV-BA distance. These cardiac morphological abnormalities are similar to the toxic effects of other pollutants, such as prothioconazole [32], triadimefon [33], and pyrimethanil [34]. A correctly looping heart is critical for the formation of a normal cardiac structure [35]. Previous reports indicated that HR is a key parameter in evaluating heart function [32, 36]. Our results showed that NaF exposure decreased the HR at 96 hpf and 120 hpf. These results suggested that NaF decreases the HR at the late stage of development. Overall, we hypothesized that NaF exposure leads to cardiac malformation in zebrafish.

Our results suggested that NaF might be toxic to heart development. As the NaF concentration increased, the severity of heart teratogenicity increased. However, little study has explored the mechanism of NaF-induced cardiotoxicity. To explore potential environmental health risks and mechanism induced by NaF, we conducted RNA-seq analysis in zebrafish larvae. In zebrafish, cardiac morphogenesis is regulated by critical genes, including Gata4, Tbx5a, Hand2, Tnnt2c, Nppa, and Myh6. In cardiac morphogenesis, Gata4 plays a critical role [37] and is a key gene in regulating cardiomyocyte proliferation and cardiac septal development [38]. The expression level of Gata4 and the distance of SV-BA were significantly increased in the NaF-treated groups. It is suggested that NaF-treated may lead to abnormal cardiac septal development. Transcription factor Tbx5a is vital for cardiac morphogenesis [39]. In zebrafish, the cardiac phenotype of Tbx5a deficiency was similar to Holt-Oram syndrome [40]. Interestingly, hyperactivity of Tbx5a also causes cardiac defects [41]. Transcription factor Hand2 is an important regulator of embryonic cardiac development and promotes cardiac reprogramming and ventricular cardiomyocyte expansion [42, 43]. The precise expression level of Hand2 is essential for normal heart morphogenesis and function [44, 45]. Cardiac troponin T (Tnnt2c) regulates the interaction between myosin and actin, and plays an important role in myofbrillogenesis in zebrafish [46, 47]. Atrial natriuretic peptide (Nppa) expression in the early-developing heart shows a restricted and dynamic pattern [48]. Nppa is a key and sensitive marker for the developing heart. For example, the patterning of Nppa expression is altered in congenital heart disease [49]. In addition, the upregulation of Nppa expression causes cardiac deformity [50]. In the developing heart, Myh6 is highly expressed and mutations of Myh6 lead to atrial septal defects and cardiomyopathy [51]. Our results showed that the expression level of the critical genes (Gata4, Tbx5a, Hand2, Tnnc2, Nppa, and Myh6) were significantly increased in the NaF-treated groups, indicating that NaF exposure causes cardiac dysmorphogenesis through these key genes.

In addition, we used GO and KEGG to analyse the potential mechanisms of cardiac deformity caused by NaF exposure. The results of bioinformatic analysis offer novel insights into the underlying mechanism. After exposure to NaF, activation of the Ras signaling pathway was detected in the 120 hpf zebrafish, indicating that NaF might lead to heart development deformity through Ras signaling. Both the Ras and Rho (RhoA, Rac1, and Cdc42) subfamilies belong to the small G protein superfamily, which regulates many cellular responses, and overexpression of RhoA promotes lethal heart failure. Rac1 and Cdc42 regulate the actin/myosin cytoskeleton in many cells [52]. Our results showed that the expression level of Cdc42l was increased in the NaF exposure group at 120 hpf, further suggesting that NaF activates the Ras signaling pathway.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway can transduce numerous of cytokines, such as tumor necrosis factor (TNF), interleukin (IL) 1?, IL-6, and transforming growth factor beta (TGF?), and then regulates a series of physiological and pathophysiological processes, including cell differentiation, cell proliferation and apoptosis, immune responses, and inflammation [53]. Our results showed that the expression level of Stat3 was significantly increased in the NaF-treated groups. This suggests that NaF activates inflammatory signaling pathways. IL-6 might mediate heart failure by activating the JAK/STAT signaling pathway. Suppressor of cytokine signaling (SOCS) regulates the JAK/STAT signaling pathway, and their interaction with other inflammatory factors might lead to heart failure. IL-6-like cytokines, JAK/STAT signaling, and SOCS play key roles in the regulation of heart failure [54]. In this study, the expression level of Socs3b was significantly increased in the NaF-treated groups, it is further indicated that activation the JAK/STAT pathway play a critical role in NaF-induced cardiac deformity.

Complement cascade and blood coagulation are two major contributors to the first line of defense against infection. The complement system plays a critical role in innate and adaptive immunity [55]. In addition to the detection and removal of pathogens [56], the complement system also participates in many other life activities, such as the removal of immune complexes, the promotion of angiogenesis, tissue regeneration, and lipid metabolism [57]. Therefore, the abnormal activation and expression of the complement system in the body will unbalance homeostasis, resulting in a variety of autoimmune and inflammation-related diseases [58]. It was reported that the complement and coagulation cascade pathway is associated with the risk of heart failure, myocardial infarction, and cardiovascular-related mortality in patients with established coronary heart disease [59]. The above studies suggest that the complement and coagulation cascade pathway play a critical role in cardiovascular disease by mediating inflammation. Our results showed that the complement and coagulation cascade pathway was the most enriched pathway in the NaF-treated group. This indicated that NaF might induce cardiac malformation through the complement and coagulation cascade pathway. In the present study, the expression levels of c3a.3, c5, c6, and c7b were significantly increased in the NaF exposure group as a result of the strong immune toxicity of NaF. The expression levels of f3a, f5, f9b, and f2r were significantly increased in the NaF exposure group. The F2r gene, also known as par1, contributes to inflammatory responses. Studies indicated that Par1 activation increased inflammation [60]. To sum up, we suspected that these factors could affect the autoimmune response of zebrafish, leading to inflammation and apoptosis, and thus might initiate the sequential process of heart development.

Conclusion

In conclusion, this study found that NaF exposure induces numerous structural and functional malformations during zebrafish development. Our comprehensive analysis of DEGs and their associated enriched pathways revealed a complicated relationship between the effects on the immune system and the cardiac system. NaF is excessively used and frequently detected; therefore, a more comprehensive understanding of its toxic effects is important to improve the risk assessment and supervision of such environmental pollution. This study provides some clues for the clinical prevention and treatment of patients with fluorosis.

Data Availability

No datasets were generated or analyzed during the current study.

References

  1. Jianjie C, Wenjuan X, Jinling C (2016) Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio). Aquat Toxicol 171:48–58

    Article  PubMed  Google Scholar

  2. Li M, Cao J, Chen J et al (2016) Waterborne fluoride exposure changed the structure and the expressions of steroidogenic-related genes in gonads of adult zebrafish (Danio rerio). Chemosphere 145:365–375

    Article  PubMed  CAS  Google Scholar

  3. Camargo JA (2003) Fluoride toxicity to aquatic organisms: a review. Chemosphere 50(3):251–264

    Article  PubMed  Google Scholar

  4. DeyBhowmik A, Shaw P, Mondal P et al (2021) Calcium and vitamin D supplementation effectively alleviates dental and skeletal fluorosis and retain elemental homeostasis in mice. Biol Trace Elem Res 199(8):3035–3044

    Article  CAS  Google Scholar

  5. Adali MK, Varol E, Aksoy F et al (2013) Impaired heart rate recovery in patients with endemic fluorosis. Biol Trace Elem Res 152(3):310–315

    Article  PubMed  CAS  Google Scholar

  6. Perumal E, Paul V, Govindarajan V et al (2013) A brief review on experimental fluorosis. Toxicol Lett 223(2):236–251

    Article  PubMed  CAS  Google Scholar

  7. Fihmonov SN, Panev NI, Korotenko OY et al (2016) Evaluation of risk factors in atherosclerosis in workers with chronic fluorine intoxication. Med Tr Prom Ekol 5:6–11

    Google Scholar

  8. Yuan ZW (1990) A study of left ventricular function of workers exposed to fluorine. Zhonghua Yu Fang Yi Xue Za Zhi [Chin J Prevent Med] 24(4):217–219

    PubMed  CAS  Google Scholar

  9. Flora SJ, Pachauri V, Mittal M et al (2011) Interactive effect of arsenic and fluoride on cardio-respiratory disorders in male rats: possible role of reactive oxygen species. Biometals: Int J Role Metal Ions Biol Biochem Med 24(4):615–628

    Article  CAS  Google Scholar

  10. Yan X, Dong N, Hao X et al (2019) Comparative transcriptomics reveals the role of the toll-like receptor signaling pathway in fluoride-induced cardiotoxicity. J Agric Food Chem 67(17):5033–5042

    Article  PubMed  CAS  Google Scholar

  11. Kiyuna LA, Albuquerque RPE, Chen CH et al (2019) Targeting mitochondrial dysfunction and oxidative stress in heart failure: challenges and opportunities. Free Radic Biol Med 129:155–168

    Article  Google Scholar

  12. Wang HW, Liu J, Zhao J et al (2018) Ca(2+) metabolic disorder and abnormal expression of cardiac troponin involved in fluoride-induced cardiomyocyte damage. Chemosphere 201:564–570

    Article  PubMed  CAS  Google Scholar

  13. Xie J, Yan X, Xu G et al (2020) ITRAQ-based proteomics reveals the potential mechanism of fluoride-induced myocardial contraction function damage. Ecotoxicol Environ Saf 197:110605

    Article  PubMed  CAS  Google Scholar

  14. Driever W, Solnica-Krezel L, Schier AF et al (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123:37–46

    Article  PubMed  CAS  Google Scholar

  15. Han L, Xia Q, Zhang L et al (2019) Induction of developmental toxicity and cardiotoxicity in zebrafish embryos/larvae by acetyl-11-keto-beta-boswellic acid (AKBA) through oxidative stress. Drug Chem Toxicol 1–8. https://doi.org/10.1080/01480545.2019.1663865

  16. Gu J, Wang H, Zhou L et al (2020) Oxidative stress in bisphenol AF-induced cardiotoxicity in zebrafish and the protective role of N-acetyl N-cysteine. Sci Total Environ 731:139190

    Article  PubMed  CAS  Google Scholar

  17. Liu X, Gao Q, Feng Z et al (2021) Protective effects of spermidine and melatonin on deltamethrin-induced cardiotoxicity and neurotoxicity in zebrafish. Cardiovasc Toxicol 21(1):29–41

    Article  PubMed  Google Scholar

  18. Molina B, Chavez J, Grainger S (2020) Zebrafish models of acute leukemias: current models and future directions. Wiley Interdiscip Rev Dev Biol e400. https://doi.org/10.1002/wdev.400

  19. Antkiewicz DS, Burns CG, Carney SA et al (2005) Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol Sci: Off J Soc Toxicol 84(2):368–377

    Article  CAS  Google Scholar

  20. Chen Z, Huang W, Dahme T et al (2008) Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms. Cardiovasc Res 79(1):97–108

    Article  PubMed  CAS  Google Scholar

  21. Wu Y, Zhang Y, Chen M et al (2019) Exposure to low-level metalaxyl impacts the cardiac development and function of zebrafish embryos. J Environ Sci 85:1–8

    Article  CAS  Google Scholar

  22. Fishman MC, Chien KR (1997) Fashioning the vertebrate heart: earliest embryonic decisions. Development 124(11):2099–2117

    Article  PubMed  CAS  Google Scholar

  23. Stainier DY, Lee RK, Fishman MC (1993) Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development 119(1):31–40

    Article  PubMed  CAS  Google Scholar

  24. Stainier DY (2001) Zebrafish genetics and vertebrate heart formation. Nat Rev Genet 2(1):39–48

    Article  PubMed  CAS  Google Scholar

  25. Thisse C, Zon LI (2002) Organogenesis–heart and blood formation from the zebrafish point of view. Science 295(5554):457–462

    Article  PubMed  CAS  Google Scholar

  26. Li H, Trager LE, Liu X et al (2022) lncExACT1 and DCHS2 regulate physiological and pathological cardiac growth. Circulation 145(16):1218–1233

    Article  PubMed  PubMed Central  CAS  Google Scholar

  27. Hong X, Zhang L, Zha J (2022) Toxicity of waterborne vortioxetine, a new antidepressant, in non-target aquatic organisms: from wonder to concern drugs? Environ Pollut 304:119175

  28. Lu J, Wang W, XU W et al (2022) Induction of developmental toxicity and cardiotoxicity in zebrafish embryos by Emamectin benzoate through oxidative stress. Sci Total Environ 825:154040

  29. Beis D, Bartman T, Jin SW et al (2005) Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development. Development 132(18):4193–4204

    Article  PubMed  CAS  Google Scholar

  30. Sipes NS, Padilla S, Knudsen TB (2011) Zebrafish: as an integrative model for twenty-first century toxicity testing. Birth Defects Res C Embryo Today 93(3):256–267

    Article  PubMed  CAS  Google Scholar

  31. He L, Chen Y, Hu Z et al (2021) Evaluation of 3,4,4,9-trichlorocarbanilide to zebrafish developmental toxicity based on transcriptomics analysis. Chemosphere 278:130349

  32. Sun Y, Cao Y, Tong L et al (2020) Exposure to prothioconazole induces developmental toxicity and cardiovascular effects on zebrafish embryo. Chemosphere 251:126418

  33. Liu HC, Chu TY, Chen LL et al (2017) The cardiovascular toxicity of triadimefon in early life stage of zebrafish and potential implications to human health. Environ Pollut 231(Pt 1):1093–1103

    Article  PubMed  CAS  Google Scholar

  34. Meng Y, Zhong K, Xiao J et al (2020) Exposure to pyrimethanil induces developmental toxicity and cardiotoxicity in zebrafish. Chemosphere 255:126889

  35. Ramasubramanian A, Nerurkar NL, Achtien KH et al (2008) On modeling morphogenesis of the looping heart following mechanical perturbations. J Biomech Eng 130(6):061018

    Article  PubMed  Google Scholar

  36. Thompson WA, Shvartsburd Z, Vijayan MM (2022) The antidepressant venlafaxine perturbs cardiac development and function in larval zebrafish. Aquat Toxicol 242:106041

  37. Holtzinger A, Evans T (2005) Gata4 regulates the formation of multiple organs. Development 132(17):4005–4014

    Article  PubMed  CAS  Google Scholar

  38. Ang YS, Rivas RN, Ribeiro AJS et al (2016) Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis. Cell 167(7):1734–49 e22

  39. Ahn DG, Kourakis MJ, Rohde LA et al (2002) T-box gene tbx5 is essential for formation of the pectoral limb bud. Nature 417(6890):754–758

    Article  PubMed  CAS  Google Scholar

  40. Garrity DM, Childs S, Fishman MC (2002) The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome. Development 129(19):4635–4645

    Article  PubMed  CAS  Google Scholar

  41. Tong X, Zu Y, Li Z et al (2014) Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish. Nat Commun 5:3153

  42. Mcfadden DG, Barbosa AC, Richardson JA et al (2005) The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner. Development 132(1):189–201

    Article  PubMed  CAS  Google Scholar

  43. Song K, Nam YJ, Luo X et al (2012) Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature 485(7400):599–604

    Article  PubMed  PubMed Central  CAS  Google Scholar

  44. Vandusen NJ, Casanovas J, Vincentz JW et al (2014) Hand2 is an essential regulator for two Notch-dependent functions within the embryonic endocardium. Cell Rep 9(6):2071–2083

    Article  PubMed  PubMed Central  CAS  Google Scholar

  45. Tsuchihashi T, Maeda J, Shin CH et al (2011) Hand2 function in second heart field progenitors is essential for cardiogenesis. Dev Biol 351(1):62–69

    Article  PubMed  CAS  Google Scholar

  46. Huang W, Zhang R, Xu X (2009) Myofibrillogenesis in the developing zebrafish heart: a functional study of tnnt2. Dev Biol 331(2):237–249

    Article  PubMed  PubMed Central  CAS  Google Scholar

  47. Parmacek MS, Solaro RJ (2004) Biology of the troponin complex in cardiac myocytes. Prog Cardiovasc Dis 47(3):159–176

    Article  PubMed  CAS  Google Scholar

  48. Zeller R, Bloch KD, Williams BS et al (1987) Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev 1(7):693–698

    Article  PubMed  CAS  Google Scholar

  49. Bruneau BG (2011) Atrial natriuretic factor in the developing heart: a signpost for cardiac morphogenesis. Can J Physiol Pharmacol 89(8):533–537

    Article  PubMed  CAS  Google Scholar

  50. Kim YC, Lee SR, Jeon HJ et al (2020) Acute toxicities of fluorene, fluorene-1-carboxylic acid, and fluorene-9-carboxylic acid on zebrafish embryos (Danio rerio): molecular mechanisms of developmental toxicities of fluorene-1-carboxylic acid. Chemosphere 260:127622

  51. Jin SC, Homsy J, Zaidi S et al (2017) Contribution of rare inherited and de novo variants in 2,871 congenital heart disease probands. Nat Genet 49(11):1593–1601

    Article  PubMed  PubMed Central  CAS  Google Scholar

  52. Clerk A, Sugden PH (2000) Small guanine nucleotide-binding proteins and myocardial hypertrophy. Circ Res 86(10):1019–1023

    Article  PubMed  CAS  Google Scholar

  53. Igaz P, Toth S, Falus A (2001) Biological and clinical significance of the JAK-STAT pathway; lessons from knockout mice. Inflamm Res: Off J Eur Histamine Res Soc 50(9):435–441

    Article  CAS  Google Scholar

  54. Terrell AM, Crisostomo PR, Wairiuko GM et al (2006) Jak/STAT/SOCS signaling circuits and associated cytokine-mediated inflammation and hypertrophy in the heart. Shock 26(3):226–234

    Article  PubMed  CAS  Google Scholar

  55. Song WC, Sarrias MR, Lambris JD (2000) Complement and innate immunity. Immunopharmacology 49(1–2):187–198

    Article  PubMed  CAS  Google Scholar

  56. Ricklin D, Lambris JD (2013) Complement in immune and inflammatory disorders: pathophysiological mechanisms. J Immunol 190(8):3831–3838

    Article  PubMed  CAS  Google Scholar

  57. Ricklin D, Hajishengallis G, Yang K et al (2010) Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11(9):785–797

    Article  PubMed  PubMed Central  CAS  Google Scholar

  58. Morgan BP, Harris CL (2015) Complement, a target for therapy in inflammatory and degenerative diseases. Nat Rev Drug Discovery 14(12):857–877

    Article  PubMed  CAS  Google Scholar

  59. Wang J, Zhang Y, Liu YM et al (2020) Uncovering the protective mechanism of Huoxue Anxin Recipe against coronary heart disease by network analysis and experimental validation. Biomed Pharmacother = Biomed Pharmacother 121:109655

  60. Tang X, Ma X, Cao J et al (2022) The influence of temperature on the antiviral response of mIgM(+) B lymphocytes against Hirame novirhabdovirus in flounder (Paralichthys olivaceus). Front Immunol 13:802638

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Funding

This work was supported by the National Natural Science Foundation of China (grant numbers 82060079, 81670290); the Natural Science Foundation of Guizhou Province (grant numbers QianKeHe Support [2022]181, ZK(2021)357, Qiankehe Cooperation Platform Talents [2021] Postdoctoral Station 007); the Research Project of Education Department of Guizhou Province (grant number QianJiaoJi [2023]037, QianJiaoJi (2022)219); the Natural Science Foundation of Guiyang City (grant numbers [2019]9–2, [2022]4–3-2, [2022]4–3-10); and the Subject Excellent Reserve Talent Project (grant number gyfyxkrc-2023–14). The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.

Author information

Author notes

  1. Feiqing Wang, Fa Chen, and Wen Song contributed equally to this work.

Authors and Affiliations

  1. Clinical Research Center, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, No. 71 Bao Shan North Road, Yunyan District, Guiyang, 550001, Guizhou Province, China

    Feiqing Wang, Fa Chen, Wen Song, Haiyan Wu, Tingting Tian, Mengxian Tian, Dongxin Tang & Yang Liu

  2. Academy of Medical Engineering and Translational Medicine, Tianjin University, Tianjin City, 300072, China

    Feiqing Wang

  3. Department of Hematology, Affiliated Hospital of Guizhou Medical University, Guiyang, 550004, Guizhou Province, China

    Yanju Li

Contributions

WFQ, LY, TDX, FC, and WS conceived of and designed the study. With full access to all data, they take responsibility for the integrity of the data, the accuracy of the data analysis, and the writing of the report. YJL, TTT, and TMX critically revised the report. HYW, YJL, and TTT performed the statistical analyses. All the authors contributed to the data acquisition and analyses. All the authors have reviewed and approved of the final version of the manuscript.

Corresponding authors

Correspondence to Dongxin Tang or Yang Liu.

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Competing Interests

The authors declare no competing interests.

Supplementary Information