A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This study primarily introduces the application of liver metabolomics in investigating the effectiveness of Shi-Liu-Bu-Xue Syrup in treating anemia.

Abstract

As a well-known Uyghur medicine, Shi-Liu-Bu-Xue Syrup (SLBXS) has been widely used to treat anemia in China for over 20 years. However, the underlying mechanisms of its effectiveness in treating anemia remain unclear. In this study, liver metabolomics was primarily employed to determine the potential regulatory mechanisms of SLBXS in treating anemia. Liver metabolomics profiling was conducted to characterize the mechanism of action of SLBXS in an acetylphenylhydrazine-induced mouse model of anemia. SLBXS was shown to decrease liver index, white blood cell count, and platelet count, while increasing red blood cell count, hemoglobin, and hematocrit levels. Core targets were selected for verification using Western blotting. SLBXS demonstrated a significant therapeutic effect on anemia primarily by regulating galactose metabolism and the HIF-1 signaling pathway, as indicated by the downregulation of HIF-1α, NOS3, VEGFA, and GLA proteins in the liver tissues of anemic mice. This study clarifies the potential regulatory mechanisms of hepatic metabolism by SLBXS administration in treating anemia.

Introduction

Anemia is a pressing and prevalent global health issue, affecting 25% of the world's population and people of all ages, particularly adolescents and pregnant women1,2,3. It is associated with an increased risk of preterm labor and maternal mortality and can lead to physical developmental disorders and impaired cardiovascular performance4. This condition may also negatively impact the health status of adolescents, resulting in infections and heart failure5. Current treatments primarily include blood transfusion, iron supplementation, and erythropoietin therapy. However, these treatments have disadvantages and adverse side effects, such as anaphylaxis, gastrointestinal upset, iron overload, and hives1. Therefore, identifying effective drugs with fewer side effects for treating anemia is crucial.

Traditional Chinese medicine, including Uyghur medicine, offers several advantages, such as multi-ingredient formulations, multi-target effects, multi-link interactions, and fewer side effects in preventing and treating multifactorial diseases. Shi-Liu-Bu-Xue Syrup (SLBXS) is a notable traditional agent in Uyghur medicine used for blood tonics and blood production. It is recognized as a blood-regulating drug that can reduce liver heat and has been included in the guidelines for the clinical use of minority medicines for treating anemia. It is also licensed by the Chinese State Food and Drug Administration (Z20026094)6,7,8. Over the past two decades, SLBXS has been extensively used in China to treat anemia-related conditions. However, its potential mechanisms for treating anemia remain unknown and require further investigation. Metabolomics, which examines the dynamic metabolic responses of biological systems to disease, drug interventions, or environmental conditions9, is increasingly used to elucidate the mechanisms of action of traditional Chinese medicine by evaluating changes in metabolic biomarkers in biological samples following external stimuli9,10.

Accordingly, a liver metabolomics approach was adopted in this study to determine the underlying therapeutic mechanisms of SLBXS in treating anemia. First, an acetylphenylhydrazine (APH)-induced mouse model of anemia was established. Next, the metabolic pathways of endogenous metabolites were investigated using liver metabolomics with gas chromatography-mass spectrometry (GC-MS) and multivariate data methods following SLBXS administration. Finally, key targets were analyzed experimentally to elucidate the anti-anemic effects and molecular mechanisms of SLBXS.

Protocol

All experimental procedures were approved by the Laboratory Animal Ethics Committee of the Hubei University of Chinese Medicine (HBUCMS201912015). Male C57BL/6 mice (weight 20-22 g) were housed in a specific pathogen-free room with a relative humidity of 50%-60% and a temperature of 22 °C ± 2 °C, subjected to a 12 h light/12 h dark cycle, and provided with free access to food and water. Before the experiment began, all mice were allowed one week to acclimate to the environment. Mice were randomly assigned to one of the following four groups (n = 12): control, model, Fu-Fang-E-Jiao Syrup (FFEJS, a positive drug, administered intragastrically at 7.8 mL/kg), and SLBXS (administered intragastrically at 11.7 mL/kg). Mice in the control and model groups received equal volumes of saline. Mice in all groups were given intragastric administration of the corresponding drugs once daily for 2 weeks. Details of the drugs, reagents, and equipment used in this study are listed in the Table of Materials.

1. Establishment of anemia model in mice

  1. Weigh 2 g of acetylphenylhydrazine (APH) using an electronic balance and transfer it into a 150 mL beaker. Add 100 mL of saline and stir with a glass rod until the APH is fully dissolved.
  2. Establish the mouse model of anemia by subcutaneously injecting 2% APH as prepared in step 1.1 on the 1st, 4th, and 7th days at dosages of 200 mg/kg, 100 mg/kg, and 100 mg/kg, respectively11.
    NOTE: Starting on the first day, mice in the FFEJS (7.8 mL/kg) and SLBXS (11.7 mL/kg) groups were given intragastric administration once daily for 2 weeks. Mice in the control and model groups received equal volumes of saline once daily for 2 weeks.

2. Determining the liver index

  1. At the end of the experiment, weigh each mouse using an electronic scale.
  2. Anesthetize the mice by inhaling 2% isoflurane. Squeeze the eyeball of the mice to make it hyperemic and protruding. Quickly remove the eyeball with forceps and collect the blood in heparinized sample tubes.
  3. Secure the anesthetized mice from step 2.2 to a surgical manipulation plate.
  4. Make a complete incision along the midline of the abdomen with a scalpel. Carefully dissect and isolate the intact liver tissue12, then measure its weight with an electronic balance.
    NOTE: The liver index of each mouse is calculated using the following formula: Liver index = liver weight/body weight.

3. Hematologic analysis

  1. Gently shake the tube containing the heparinized blood sample from step 2.2 to prevent blood clotting.
  2. Place the blood sample below the injection needle to ensure that it is fully immersed in the needle.
  3. Click the Auto Detect button to measure red blood cell count (RBC), hematocrit (HCT), white blood cell count (WBC), hemoglobin (HGB) levels, and platelet count (PLT) using a fully automatic hemocyte analyzer.

4. Liver metabolomics study

  1. Liver sample preparation
    1. Homogenize liver tissue samples (50 mg) from step 2.4 with 1 mL of pre-chilled methanol. Centrifuge at 18,759 x g for 10 min at 4 °C to remove the precipitate.
    2. Transfer 200 µL of the supernatant to a sample vial using a pipette and vacuum-dry in a freeze-drier at 35 °C for 2 h.
    3. React the dried samples with 40 µL of a 40 mg/mL solution of methoxyamine hydrochloride in pyridine for 90 min at 30 °C. Then, add 80 µL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% Trimethylchlorosilane (TMCS) and incubate for 60 min at 37 °C.
    4. Add 10 µL of n-hexane to the vial to terminate the derivatization reaction.
  2. Liver metabolic analysis
    1. Analyze the derivatized samples (1 µL) using a GC-MS system. Separate the derivatives using a DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm).
      NOTE: The oven temperature program conditions were set as follows: 60 °C for 1 min; increase to 325 °C at a rate of 10 °C/min and maintain for 10 min. The injector, ion source, and MS temperatures were set to 250 °C, 230 °C, and 150 °C, respectively. Helium (99.999%) was used as the carrier gas at a flow rate of 1.1 mL/min, and the split ratio was set to 10:1. An electron beam energy of 70 eV and a solvent delay time of 5.9 min were used.
  3. Data processing and analysis
    1. Acquire and convert raw GC-MS data using the compatible MassHunter software.
    2. Conduct spectral analysis using the Automated Mass Spectral Deconvolution and Identification System (AMDIS) tool12.
    3. Identify all metabolites using the NIST and HMDB databases (see Table of Materials).
    4. Import the data into the MetaboAnalyst tool for partial least squares-discriminant analysis (PLS-DA), t-tests, pathway analysis, and network analysis12.

5. Western blot analysis

  1. Extract the total proteins from mouse liver tissue
    1. Add 50 mg of liver tissue from step 2.4 and 250 µL of cell lysate to a 1 mL glass homogenizer and grind on ice for 5 min.
    2. Transfer the liver tissue homogenate from step 5.1.1 to a 1.5 mL microcentrifuge tube using a pipettor, and centrifuge at 18,759 x g for 10 min at 4 °C. Then transfer the supernatant to a new 1.5 mL tube using a pipettor.
  2. Determine protein concentrations and pre-process protein samples
    1. Add 2 µL of supernatant from step 5.1.2, 18 µL of PBS, and 180 µL of BCA working solution to a 96-well microtiter plate8.
    2. Oscillate the plate on an oscillator for 30 s, leave it for 30 min at 37 °C, and determine the absorbance at 562 nm using a microplate reader.
  3. Separate the total proteins using SDS-PAGE, transfer to polyvinylidene fluoride membranes, and block with 5% nonfat milk8.
  4. Incubate the membranes from step 5.3 with primary antibodies against HIF-1α (1:1000), VEGFA (1:1000), GLA (1:1000), NOS3 (1:1000), and β-actin (1:5000) overnight at 4 °C.
  5. Place the membranes from step 5.4 in an antibody incubation box, add 10 mL of TBST, and horizontally shake at 111 x g at room temperature to wash off unbound primary antibodies three times for 5 min each.
  6. Add 200 µL of goat anti-rabbit IgG (H + L)-HRP (1:1000) to each membrane from step 5.5 and incubate for 2 h at room temperature. Then, repeat step 5.5 to wash off unbound secondary antibody (goat anti-rabbit IgG (H + L)-HRP).
  7. Add 200 µL of ultrahigh sensitivity ECL chemiluminescent solution to the surface of each membrane from step 5.6 and immediately visualize protein bands using an automatic chemiluminescence imaging analysis system.

6. Statistical analysis

  1. Analyze the data using statistical and graphing software with one-way ANOVA followed by Tukey's test.
  2. Present the results as mean ± standard deviation (SD) and consider a P-value < 0.05 as statistically significant.

Results

To confirm the successful establishment of the mouse model of anemia and analyze the effect of SLBXS on anemia, the liver index and hematological parameters were first investigated. Figure 1 illustrates that the model group exhibited a significant decrease (P < 0.01) in red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) compared to the control group. Conversely, the liver index, white blood cell count (WBC), and platelet count (PLT) in the model group were notabl...

Discussion

Anemia is a common condition affecting many people worldwide, particularly in developing countries1. In China, patients frequently use traditional Chinese medicine, including Uyghur medicine, to alleviate the signs and symptoms of anemia. SLBXS is a Uyghur medicine that has been used in clinical practice for many years; however, its exact mechanism of action against anemia remains poorly understood13. In this study, a mouse model of acetylphenylhydrazine (APH)-induced anemi...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Special Training Plan for Minority Science and Technology Talents, Natural Science Foundation of Xinjiang Uyghur Autonomous Region (2020D03021), the Funds for Key Program for Traditional Chinese Medicine of Hubei University of Chinese Medicine (2022ZZXZ004), and Tianshan Innovation Team Program (2020D14030).

Materials

NameCompanyCatalog NumberComments
AcetylphenylhydrazineShanghai Aladdin Biochemical Technology Co., Ltd.C13979660
Automatic chemiluminescence imaging analysis systemShanghai Tanon Life Science Co., Ltd.Tanon-5200
Bicinchoninic acid assay kitThermoFisher ScientificQPBCA-1KT
Capillary columnAgilent J&W Scientific, Agilent Technologies, Inc.DB-5MS
Cell lysis buffer for Western and IPBeyotime BiotechnologyP0013
ChlorotrimethylsilaneShanghai Aladdin Biochemical Technology Co., Ltd.C104814
Electronic balanceMettler-Toledo International Inc.ME203E
Electronic scaleMettler-Toledo International Inc.LE104E
Fu-Fang-E-Jiao SyrupDong E E Jiao Co., Ltd.214020031
Fully automatic hemocyte analyzerShenzhen Mindray Animal Care Technology Co., Ltd.IDEXX ProCyte Dx
GC-MS systemAgilent Technologies, Inc.7890B-5977B 
GLA primary antibodyBioworld TechnologyBS77041
Glass homogenizerShanghai Lei Gu Instruments Co., Ltd.B-013001
Glass rod Shanghai Lei Gu Instruments Co., Ltd.B-003904
GraphPad Prism software GraphPad, La JollaVersion 9.0
Heparinized sample tubesChangde BKMAM Biotechnology Co., Ltd.B-ACT1P5
HIF-1α primary antibodyBioworld TechnologyBS3514
HMDB databasehttp://www.hmdb.ca/—
IsofluraneHebei Jindafu Pharmaceutical Co., Ltd.20231202
Male C57BL/6 miceLiaoning Changsheng Biotechnology Co., Ltd.No. SCXK [Liao] 2015-0001
MassHunterAgilent Technologies, Inc.B.08.00
MetaboAnalyst 5.0https://www.metaboanalyst.ca/—
Methoxyamine hydrochlorideShanghai Aladdin Biochemical Technology Co., Ltd.E1818113
n-hexaneShanghai Aladdin Biochemical Technology Co., Ltd.C14878803
NIST databasehttp://webbook.nist.gov/chemistry/—
NOS3 primary antibodyBioworld TechnologyBS3625
PyridineShanghai Aladdin Biochemical Technology Co., Ltd.C13026996
SalineBIOSHARP LIFE SCIENCES2308262009
Shi-Liu-Bu-Xue SyrupXinjiang Uygur Pharmaceutical Co., Ltd.211277
Surgical manipulation plateDIXSGZK-JPB-A
VEGFA primary antibodyBioworld TechnologyAP0742
β-actinABclonal (Shanghai) Trading Co., Ltd.AC026

References

  1. Munoz, M., Gomez-Ramirez, S., Bhandari, S. The safety of available treatment options for iron-deficiency anemia. Expert Opin Drug Saf. 17 (2), 149-159 (2018).
  2. Soliman, A. T., De Sanctis, V., Yassin, M., Wagdy, M., Soliman, N. Chronic anemia and thyroid function. Acta Biomed. 88 (1), 119-127 (2017).
  3. Pivina, L., Semenova, Y., Dosa, M. D., Dauletyarova, M., Bjorklund, G. Iron deficiency, cognitive functions, and neurobehavioral disorders in children. J Mol Neurosci. 68 (1), 1-10 (2019).
  4. Lumbiganon, P., et al. Indirect causes of severe adverse maternal outcomes: a secondary analysis of the WHO Multicountry Survey on Maternal and Newborn Health. BJOG. 121 (Suppl 1), 32-39 (2014).
  5. Shaddy, R. E., et al. Systematic literature review on the incidence and prevalence of heart failure in children and adolescents. Pediatr Cardiol. 39 (3), 415-436 (2018).
  6. China Medical Association of Minorities. . Guidelines for clinical use of minority drugs. , (2018).
  7. Yu, W. Q. Clinical efficiency of Pomegranate blood-enriching syrup in the treatment of iron deficiency anemia. J Med Pharm Chin Minor. 26 (11), 11-12 (2020).
  8. Zhang, D. N., et al. Mechanism of Shiliu Buxue Syrup for anemia using integrated metabolomics and network pharmacology. Anal Biochem. 653, 114774 (2022).
  9. Nielsen, J. Systems biology of metabolism. Annu Rev Biochem. 86, 245-275 (2017).
  10. Wang, M., et al. Metabolomics highlights pharmacological bioactivity and biochemical mechanism of traditional Chinese medicine. Chem Biol Interact. 273, 133-141 (2017).
  11. Ben, C. N. Establishment and experimental research of "Blood Deficiency" animal model. Lab Animal Sci. (03), 5-10 (1994).
  12. Cao, S., et al. Integrative transcriptomics and metabolomics analyses provide hepatotoxicity mechanisms of asarum. Exp Ther Med. 20 (2), 1359-1370 (2020).
  13. Yang, H. Y., Liu, M. L., Luo, P., Yao, X. S., Zhou, H. Network pharmacology and metabolomics in the study of traditional Chinese medicine for cardiovascular diseases. Phytomedicine. 104, 154268 (2022).
  14. Croci, S., Pedrazzi, G., Passeri, G., Piccolo, P., Ortalli, I. Acetylphenylhydrazine induced haemoglobin oxidation in erythrocytes studied by Mossbauer spectroscopy. Biochim Biophys Acta. 1568 (1), 99-104 (2001).
  15. Liebowitz, J., Cohen, G. Increased hydrogen peroxide levels in glucose 6-phosphate dehydrogenase deficient erythrocytes expose to acetylphenylhydrazine. Biochem Pharmacol. 17 (6), 983-988 (1968).
  16. Tarasenko, T. N., McGuire, P. J. The liver is a metabolic and immunologic organ: A reconsideration of metabolic decompensation due to infection in inborn errors of metabolism (IEM). Mol Genet Metab. 121 (4), 283-288 (2017).
  17. Topalovic, A., Knezevic, M., Gacnik, S., Mikulic-Petkovsek, M. Detailed chemical composition of juice from autochthonous pomegranate genotypes (Punica granatum L.) grown in different locations in Montenegro. Food Chem. 330, 127261 (2020).
  18. Legua, P., et al. Total phenols and antioxidant capacity in 10 Moroccan pomegranate varieties. J Food Sci. 77 (1), C115-C120 (2012).
  19. Rodrigo, R., Miranda, A., Vergara, L. Modulation of endogenous antioxidant system by wine polyphenols in human disease. Clin Chim Acta. 412 (5-6), 410-424 (2011).
  20. Hussain, T., et al. Oxidative stress and inflammation: What polyphenols can do for us. Oxid Med Cell Longev. 2016, 7432797 (2016).
  21. Sudheesh, S., Vijayalakshmi, N. R. Flavonoids from Punica granatum--potential antiperoxidative agents. Fitoterapia. 76 (2), 181-186 (2005).
  22. Riaz, A., Khan, R. A. Anticoagulant, antiplatelet and antianemic effects of Punica granatum (pomegranate) juice in rabbits. Blood Coagul Fibrinolysis. 27 (3), 287-293 (2016).
  23. Balasubramani, S. P., Varghese, R. K., Vishnuprasad, C. N., Venkatasubramanian, P. Pomegranate juice enhances iron dialysability and assimilation in in-vitro cell-free and cell-based models. Plant Foods Hum Nutr. 75 (2), 272-278 (2020).
  24. Jilani, T., Iqbal, M. P. Does vitamin E have a role in treatment and prevention of anemia. Pak J Pharm Sci. 24 (2), 237-242 (2011).
  25. Farrell, P. M., Bieri, J. G., Fratantoni, J. F., Wood, R. E., di Sant'Agnese, P. A. The occurrence and effects of human vitamin E deficiency. A study in patients with cystic fibrosis. J Clin Invest. 60 (1), 233-241 (1977).
  26. Ukab, W. A., Sato, J., Wang, Y. M., van Eys, J. Xylitol mediated amelioration of acetylphenylhydrazine-induced hemolysis in rabbits. Metabolism. 30 (11), 1053-1059 (1981).
  27. Ahuja, V., et al. Biological and pharmacological potential of xylitol: A molecular insight of unique metabolism. Foods. 9 (11), 1592 (2020).
  28. Nikas, I. P., Paschou, S. A., Ryu, H. S. The role of nicotinamide in cancer chemoprevention and therapy. Biomolecules. 10 (3), 477 (2020).
  29. Katayoshi, T., Yamaura, N., Nakajo, T., Kitajima, N., Tsuji-Naito, K. Porcine placental extract increases the cellular NAD levels in human epidermal keratinocytes. Sci Rep. 12 (1), 19040 (2022).
  30. Bartoszewski, R., et al. Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia. Faseb J. 33 (7), 7929-7941 (2019).
  31. Lee, S. H., Golinska, M., Griffiths, J. R. HIF-1-independent mechanisms regulating metabolic adaptation in hypoxic cancer cells. Cells. 10 (9), 2371 (2021).
  32. Sato, T., Takeda, N. The roles of HIF-1α signaling in cardiovascular diseases. J Cardiol. 81 (2), 202-208 (2023).
  33. Ratcliffe, P. J. Oxygen sensing and hypoxia signalling pathways in animals: The implications of physiology for cancer. J Physiol. 591 (8), 2027-2042 (2013).
  34. Li, L. J., Huang, Q., Zhang, N., Wang, G. B., Liu, Y. H. miR-376b-5p regulates angiogenesis in cerebral ischemia. Mol Med Rep. 10 (1), 527-535 (2014).
  35. Tejero, J., Shiva, S., Gladwin, M. T. Sources of vascular nitric oxide and reactive oxygen species and their regulation. Physiol Rev. 99 (1), 311-379 (2019).
  36. Fujii, J., et al. Erythrocytes as a preferential target of oxidative stress in blood. Free Radic Res. 55 (5), 562-580 (2021).
  37. Huang, J. W., et al. High expression of HIF-1α alleviates benzene-induced hematopoietic toxicity and immunosuppression in mice. Environ Pollut. 311, 119928 (2022).
  38. Zhang, Z., Yao, L., Yang, J., Wang, Z., Du, G. PI3K/Akt and HIF-1 signaling pathway in hypoxia-ischemia (Review). Mol Med Rep. 18 (4), 3547-3554 (2018).
  39. Del Balzo, U., et al. Nonclinical characterization of the hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat, a novel treatment of anemia of chronic kidney disease. J Pharmacol Exp Ther. 374 (2), 342-353 (2020).
  40. Bhatia, S., Singh, A., Batra, N., Singh, J. Microbial production and biotechnological applications of α-galactosidase. Int J Biol Macromol. 150, 1294-1313 (2020).
  41. Conte, F., van Buuringen, N., Voermans, N. C., Lefeber, D. J. Galactose in human metabolism, glycosylation and congenital metabolic diseases: Time for a closer look. Biochim Biophys Acta Gen Subj. 1865 (8), 129898 (2021).
  42. Wang, Y. Y. . Investigate the effect of galacto-oligosaccharide supplementation on intestinal absorption of iron in rats. [Master's Thesis]. , (2019).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Shi Liu Bu Xue SyrupAnemia TreatmentLiver MetabolomicsMechanism Of ActionAcetylphenylhydrazineTherapeutic EffectGalactose MetabolismHIF 1 Signaling PathwayWestern BlottingLiver IndexWhite Blood Cell CountRed Blood Cell CountHemoglobin LevelsMetabolic Profiling

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved