International Journal of Biological Macromolecules 277 (2024) 134450

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Caulerpa chemnitzia polysaccharide exerts immunomodulatory activity in

macrophages by mediating the succinate/PHD2/HIF-1α/IL-1β pathway
Jinzi Zeng a, 1 , Jun Liu b, 1 , Ning Zhao c , Io Nam Wong d, * , Riming Huang a, *
a
Guangdong Provincial Key Laboratory of Food Quality and Safety/College of Food Science, South China Agricultural University, Guangzhou 510642, China
b
Laboratory of Pathogenic Biology, Guangdong Medical University, Zhanjiang 524023, China
c
Shenzhen Hospital of Integrated Traditional Chinese and Western Medicine, Shenzhen 518104, China
d
Faculty of Medicine, Macau University of Science and Technology, Macau 999078, Macau

A R T I C L E I N F O A B S T R A C T

Keywords: Algal polysaccharide is an important food functional factor with diverse bioactive and low toxicity. Previous
Caulerpa chemnitzia polysaccharide studies have confirmed Caulerpa chemnitzia polysaccharides (CRVP) have immunomodulatory activity, but the
Immunomodulatory activity immunomodulatory mechanism of CRVP in macrophages has not been thoroughly explored yet. In our research,
Macrophage
we found that CRVP has outstanding immunomodulatory activity in macrophages, which is reflected in pro-
Immunometabolism
Molecular mechanism
moting cell proliferation, upregulating cytokines (IL-1β, IL-6, and TNF-α) expression, and increasing NO and ROS
levels. Additionally, the result of joint analysis of untargeted metabolomics showed metabolism played a major
role in the immunomodulatory of CRVP and suggested succinic acid was a key metabolite. Further verification
indicated that the accumulation of succinic acid in macrophages after administered with CRVP, induced the
down-regulation of prolyl hydroxylase domain 2 (PHD2) and up-regulation of hypoxia-inducible factor-1α (HIF-
1α), thereby enhancing IL-1β expression. Together, the immunomodulatory activity of CRVP in macrophages via
succinate/PHD2/HIF-1α/IL-1β pathway.

1. Introduction in the regulation of immune cell function, such as lactic acid [9], suc-
cinic acid [10], itaconic acid [11], kynurenic acid-tryptophan [12],
The immune system is a dynamic and multi-layered biological sys- GABA [13] and D-2HG [14]. Hereby, it could be a new strategy for
tem. At immune homeostasis, the immune system not only protects the preventing and controlling disease by modulating immunometabolism
body from pathogen invasion and tumor development, but also plays a to maintain immune homeostasis.
vibrant part in tissue homeostasis and organ regeneration [1]; as im- In recent years, polysaccharides have caught the attention of many
mune homeostasis is disrupted, various diseases occur and develop, such scientists due to their multiple biological activities and low toxicity
as cancer [2], autoimmune disease [3], cardiovascular disease [4], [15,16]. Caulerpa chemnitzia polysaccharide (original name was Cau-
diabetes [5], and chronic respiratory diseases [6]. Therefore, main- lerpa racemosa var. peltate, CRVP) is a natural polysaccharide extracted
taining immune homeostasis is important for the body to keep healthy. from the edible green alga Caulerpa chemnitzia by water-extraction and
Immunometabolism is an energetic field that has received a lot of alcohol-precipitation method, containing abundant sulfate moieties
attention in recent years, and it focuses on describing the changes that (25.8 % ± 0.7 %) and consisting of a large amount of mannose (92.1 %)
occur in intracellular metabolic pathways during the activation of im- [17]. Previous studies by our research team have clarified the structural
mune cells [7]. It has been demonstrated that the microenvironment characterization of CRVP-1 and preliminary evaluated the immuno-
present in the organism affects the metabolism of immune cells and even modulatory activity, which suggested that CRVP-1 boosted the prolif-
regulates the differentiation and function of immune cells, ultimately eration of RAW264.7 and the release of cytokines and NO. Furthermore,
affecting the immune homeostasis of the organism by changing the fate CRVP-1 was proved to activate the immunomodulatory in macrophage
of immune cells [8]. In the past, besides physiological functions, many RAW264.7 via TLR4/NF-κB pathway [18]. At the animal level, a study
metabolites were considered as signal transduction molecules involved showed that Caulerpa racemosa var peltate polysaccharide has positive

* Corresponding authors.
E-mail addresses: inwong@must.edu.mo (I.N. Wong), huangriming@scau.edu.cn (R. Huang).
1
These authors contributed equally to this work and share the first authorship.

https://doi.org/10.1016/j.ijbiomac.2024.134450
Received 25 April 2024; Received in revised form 12 July 2024; Accepted 1 August 2024
Available online 3 August 2024
0141-8130/© 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
J. Zeng et al. International Journal of Biological Macromolecules 277 (2024) 134450

effects on thymic index, T-cell subsets, and percentage of NK cells in 2.5. Detection of ROS
immunocompromised mice [19]. However, the mechanism research of
CRVP about immunomodulatory is still lacking. BODIPY™ 581/591 C11 (D3861, ThermoFisher Scientific, USA) was
In this paper, macrophages RAW264.7 and THP-1 were adopted to used for the determination of ROS in the cell and the cell membrane.
cell models, and the immunomodulatory activity of CRVP was first RAW264.7 and THP-1 cells were seeded in 12-well plates at 2.0 × 105/
evaluated by assessing the ability of cell proliferation, cytokines release, well. After the treatments as described above, cells were washed twice
and oxidative stress modulation. Then, metabolomics was used to with PBS, followed by incubation with 10 μM BODIPY™ 581/591 C11
explore potential metabolites to construct the possible pathway for for 1 h at 37 ◦ C in darkness, then the cells were washed three times with
CRVP to exert immunomodulatory activity from the perspective of PBS, respectively. Finally, fluorescence was measured using flow
immunometabolism. Finally, experiments were designed and molecular cytometry (CytoFLEX, Beckman Coulter, USA) and the fluorescence in-
biology tests (RT-qPCR and Western blot) were carried out to verify and tensity of each group was analyzed by using FlowJo v10.3 software.
explain the immunomodulatory molecular mechanism of CRVP in
macrophages. Thus, providing new perspectives to explain the regula- 2.6. Metabolites analysis
tion of macrophages by polysaccharides.
Metabolites analysis was performed as detailed previously [20].
2. Materials and methods Briefly, about 1 × 106 cells were collected as one biological replicate for
analysis and 3 biological replicates in each group. 2 groups were
2.1. Materials designed for both RAW264.7 and THP-1 cells, including the Control
group (CON) and CRVP treated group (CRVP). Then, metabolites were
Caulerpa chemnitzia was collected from an area in Zhanjiang, China, analyzed by the Thermo Vanquish HPLC system and Orbitrap Exploris
in April 2021. And Caulerpa chemnitzia polysaccharide was extracted 120 (ThermoFisher Scientific, USA). Chromatographic separation was
and purified according to our previously published method [17]. achieved using ACQUITY UPLC ® HSS T3 (2.1 × 100 mm, 1.8 μm)
(Waters, USA). The detailed process was described in the supplementary
2.2. Cell cultures and differentiation file. MS data were acquired using negative and positive ionization in full
scan mode over the range of m/z 100–1000. After pre-processing such as
RAW 264.7 cell line was obtained from Jinan University (Guangz- format conversion, peak identification, filtering, alignment, and
hou, China) and THP-1 cell line was purchased from Pricella Biotech- normalization, PCA, PLS-DA, and OPLS-DA were applied to data anal-
nology (Wuhan, China). RAW 264.7 cells were cultured in DMEM ysis. Finally, p < 0.05 and VIP > 1 were considered to be differently
(Pricella Biotechnology, Wuhan, China) supplemented with 10 % (v/v) accumulated metabolites (DAMs). The result was analyzed by Biodeep
FBS (Excell Biotechnology Co., Ltd., Shanghai, China) and 1 % (v/v) Cloud (https://www.biodeep.cn).
penicillin-streptomycin (Pricella Biotechnology, Wuhan, China), while
THP-1 cells were cultured in RPMI 1640 medium (Pricella Biotech- 2.7. Detection of succinite acid
nology, Wuhan, China) supplemented with 10 % (v/v) FBS and 1 % (v/
v) penicillin-streptomycin. All cells were cultivated in sterile tissue Succinite acid was quantified using LC-MS. In brief, sample pre-
culture flasks at 37 ◦ C in a humidified atmosphere containing 5 % CO2. treatment in a manner consistent with “Metabolites analysis”, and Agi-
THP-1 monocytes were differentiated into THP-1 macrophages by lent 1290-6470A (Agilent, USA) HPLC-MS system with column SB-AQ
treatment with 100 ng/mL PMA (Sigma-Aldrich, USA) for 2 days C18 (2.1 × 150 mm, 1.8 μm) (Agilent, USA) was used for detecting.
(Fig. S1), which were then used in the subsequent experiments. The detailed process was described in the supplementary file. MS data
were collected in negative ionization, and the intracellular concentra-
2.3. Detection of cell viability tion of succinic acid was calculated from a standard curve of succinic
acid (CAS: 110-15-6, Solarbio Science & Technology, Beijing, China)
The viability of macrophage RAW264.7 and THP-1 cells was concentration.
measured by using a CCK-8 assay (Yeason Biotechnology, Shanghai,
China). Briefly, macrophage RAW264.7 cells were seeded at 2 × 104 2.8. RNA extraction and real-time qPCR (RT-qPCR)
cells/well in 96-well plates and cultured in a 5 % CO2 humidified
incubator at 37 ◦ C for 24 h. CRVP samples were added at the final Total RNA was gained from cells using TRIzol Reagent (#15596026,
concentrations of 0, 1, 2.5, 5, 10, and 20 μg/mL. As for THP-1, THP-1 ThermoFisher Scientific, USA) according to the manufacturer’s in-
monocytes in 96-well plates with 2 × 104 cells per well were induced by structions. Determine the purity and concentration of RNA according to
PMA for 2 days, and then CRVP samples were added at the final con- the absorbance at 260/280 nm measured by Nanodrop 2000 spectro-
centrations of 0, 10, 20, 40, 80 and 160 μg/mL. Treated with 1 mg/mL photometer (ThermoFisher Scientific, USA). cDNA was synthesized by
LPS was used as a positive group. After incubating for 24 h, 10 μL of using the cDNA Reverse Transcription Kit for qPCR (#11141ES60,
CCK-8 solution was added to each well and incubated for 1.5 h. The Yeason Biotechnology, Shanghai, China) and then RT-qPCR was per-
absorbance of each well was measured by a microplate reader at 450 formed on the QuantStudio 3 Real-Time PCR System (Applied Bio-
nm. For cell viability assay, each concentration was repeated four times. systems, ThermoFisher Scientific, USA) using SYBR Green Master Mix
(#11184ES08, Yeason Biotechnology, Shanghai, China). The relative
2.4. Detection of cytokines and NO production mRNA expression was analyzed using the 2− ΔΔCt method and the mRNA
expression for each molecule of interest was normalized to the β-Actin
The production of IL-1β (Sangon Biotech, Shanghai, China), IL-6 expression level. The primer sequence is shown in Table S1.
(Mouse: NeoBioscience Technology, Shenzhen, China; Human: Sangon
Biotech, Shanghai, China), TNF-α (Mouse: NeoBioscience Technology, 2.9. Western blotting
Shenzhen, China; Human: Sangon Biotech, Shanghai, China) and NO
(Beyotime Biotechnology, Shanghai, China) in the supernatants of the Briefly, the samples were boiled for 10 min, electrophoresed in 10 %
macrophage RAW264.7 and THP-1 from each group were determined SDS polyacrylamide gel, and transferred onto PVDF membranes (Milli-
using commercial kits following the manufacturer’s instructions. Briefly, pore, USA). The blots were blocked with Protein Free Rapid Blocking
RAW264.7 and THP-1 were treated as described above. Subsequently, Buffer (G2052, Servicebio Biotechnology, Wuhan, China) for 10 min at
the supernatants from each group were collected for determination. room temperature and probed with GAPDH (1:10000, Affinity

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J. Zeng et al. International Journal of Biological Macromolecules 277 (2024) 134450

Biosciences, Liyang, China), HIF-1α (1:3000, Affinity Biosciences, same time, excessive concentrations may be detrimental to macrophage
Liyang, China) and PHD2 (1:2000, Affinity Biosciences, Liyang, China) proliferation. Thus, the concentration of 5, 10, and 20 μg/mL were
primary antibody at the appropriate dilutions overnight at 4 ◦ C. The selected for RAW264.7, while 20, 40, and 80 μg/mL for THP-1 in the
blots were washed and incubated for 1 h at room temperature with the following research.
HRP-conjugated secondary antibody, then developed with super ECL
detection reagents (#36222, Yeason Biotechnology, Shanghai, China)
on Amersham Imager 600 (GE HealthCare, USA). The densitometry of 3.2. CRVP promotes the release of pro-inflammatory cytokines from
protein bands was quantified using Image J software (National Institutes macrophages
of Health, USA).
The release of cytokines is an important indicator of immunomod-
ulatory activity [21]. In this study, the expression of pro-inflammatory
2.10. Experimental groups
cytokines (such as IL-1β, IL-6, and TNF-α) were determined by RT-
qPCR and Elisa kits. The results of both RT-qPCR and Elisa showed
Succinic acid-rich culture experiment. In this experiment, four
that CRVP upregulated the expression of pro-inflammatory cytokines IL-
groups were set: control (CON) group, succinic acid (SA) group, CRVP
1β, IL-6, and TNF-α in macrophages RAW264.7 and THP-1 (Figs. 2 and
(CRVP) group, and CRVP and succinic acid joint (CRVP+SA) group. The
S2). Notably, gene expressions of pro-inflammatory-related were
CON group was treated with PBS; the SA group was treated with 10 μM
increased dose-dependently after CRVP-stimulated. At the same time,
succinic acid; the CRVP group was treated with 20 μg/mL CRVP in
the cytokine levels determined with Elisa showed that a high concen-
RAW264.7 or 80 μg/mL CRVP in THP-1; and the CRVP+SA group was
tration of CRVP (80 μg/mL) might hinder the synthesis and secretion of
treated with (20 or 80 μg/mL) CRVP and 10 μM succinic acid. After 24 h
IL-6 and IL-1β in THP-1, which could be a transcriptional-translational
of cell seeding, each group was treated with corresponding methods.
conflict [22].
After incubating for 24 h at 37 ◦ C in a humidified atmosphere containing
5 % CO2., the cells were used for subsequent experiments.
Inhibitor rescue experiment. In this experiment, four groups were
set: the control (CON) group, HIF-1α inhibitor (LW6) group, CRVP 3.3. CRVP increases oxidative stress levels in macrophages
(CRVP) group, and CRVP and HIF-1α inhibitor joint (CRVP+LW6)
group. The CON group was treated with PBS; the LW6 group was treated It has been well recognized that redox-related mechanisms regulate
with 10 μM LW6 (HIF-1α inhibitor); the CRVP group was treated with immune cell function, performance, and survival, and immune-
20 μg/mL CRVP in RAW264.7 or 80 μg/mL CRVP in THP-1; and the inflammatory responses correlate with increased oxidative stress [23].
CRVP+LW6 group was treated with (20 or 80 μg/mL) CRVP and 10 μM NO is synthesized by inducible nitric oxide synthase (iNOS) that par-
LW6. Other processing methods were the same as the “Successful acid- ticipates in the invasion of pathogens and modulates the inflammatory
rich culture experience”. response, playing an important role in the immune system [24].
Meanwhile, ROS, as an oxidant, is involved in a large number of cellular
processes and contributes to the activation and regulation of the im-
2.11. Statistical analysis mune response [25]. According to Fig. 3A, CRVP significantly promoted
the release of NO in RAW264.7, While NO expression was insensitive to
All experiments were conducted three times and all data was shown the stimulation of CRVP in THP-1 (Fig. 3B). This may be related to the
as “− x ± SD”. Statistical comparisons were made using the Student’s-test characteristics of cell lines (such as the sensitivities to substances or the
or one-way analysis of variance (ANOVA) by SPSS v27 (IBM, USA), p < expression levels of genes and proteins) and the interference of THP-1
0.05 was considered statistically significant. All data were visualized macrophage inducer PMA [26,27]. Additionally, CRVP stimulation
using GraphPad Prism v9.0 (GraphPad Software, La Jolla, California, induced a boost in ROS intracellularly (Fig. 3C and D). Overall, CRVP
USA). increased the level of oxidative stress in macrophages.

3. Result
3.4. Metabolism plays a role in the immunomodulatory effects of CRVP
3.1. CRVP promotes macrophage proliferation
The results of the multivariate data analysis demonstrated that the
To determine the safe administration concentration of CRVP on CRVP group and the CON group could be distinguished and good for
macrophages, the cell viability was measured with CCK-8 assay. The prediction in macrophage RAW264.7 and THP-1 (Figs. S3 and S4). Ac-
result showed that CRVP without cytotoxicity at the treated concen- cording to VIP > 1 and p value <0.05 (Fig. 4A and B), 112 DAMs were
tration on macrophages, even promoted the proliferation of macro- filtered between the CON group and the CRVP group in RAW264.7 (120
phages compared to the control group at the range of 2.5–20 μg/mL on DAMs were up-regulated and 57 DAMs were down-regulated) (Fig. 4C,
RAW264.7 and at the range of 10–80 μg/mL on THP-1 (Fig. 1). At the Table S2). In contrast, 49 DAMs were filtered between the CON group
and the CRVP group in THP-1 (31 DAMs were up-regulated and 18
DAMs were down-regulated) (Fig. 4D, Table S3).
Next, KEGG enrichment analysis was used to explore the effects of
these DAMs in macrophages after stimulating by CRVP. The result
showed that 26 KEGG pathways were enriched significantly (p < 0.05)
in RAW264.7 (Fig. 4E, Table S4), which mainly concentrated in meta-
bolism (13 pathways, 50 %), organismal systems (5 pathways, 19.2 %),
human diseases (5 pathways, 19.2 %), and environmental information
processing (3 pathways, 11.8 %). In addition, 49 DAMs screened in THP-
1 were enriched into 19 pathways (Fig. 4F, Table S5), which mainly
concentrated in metabolism (7 pathways, 36.8 %), organismal systems
(4 pathways, 21.1 %), environmental information processing (4 path-
Fig. 1. Effect of CRVP on the proliferative capacity of macrophages RAW264.7 ways, 21.1 %), human diseases (3 pathways, 15.8 %), and genetic in-
(A) and THP-1 (B). *p < 0.05, **p < 0.01, ***p < 0.001. formation processing (1 pathway, 5.2 %).

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Fig. 2. CRVP promotes secretion of pro-inflammatory cytokines by macrophages. Secretion of IL-1β (A), IL-6 (B), and TNF-α (C) by macrophages RAW264.7.
Secretion of IL-1β (D), IL-6 (E) and TNF-α (F) by macrophages THP-1. *p < 0.05, **p < 0.01, ***p < 0.001.

3.5. Succinic acid possibly contributes to IL-1β elevation in CRVP- could regulate the expression of IL-1β by modulating HIF-α [28]. The
stimulated macrophages result of RT-qPCR showed that macrophage THP-1 culture with succinic
acid significantly up-regulated the m RNA expression of IL-1β and HIF-
Based on the above results, it is easy to observe that the pathway type 1α, the same trends were shown in CRVP group and CRVP+SA group
of “metabolism” was ranked first among all pathway types both in (Fig. 6D and F). Even though we did not observe significant changes
RAW264.7 and THP-1, which hinted metabolism possibly plays a major between the CON and SA groups at the gene expression of IL-1β and HIF-
role in CRVP involved in macrophage immunoregulation. At the same 1α in macrophage RAW264.7 (Fig. 6C and E), succinic acid affected the
time, we carried out co-analysis of metabolites screened in RAW264.7 levels of transcription of genes in a way. Notably, there was a positive
and THP-1, and the result showed that 4 DAMs were identified as correlation among the levels of succinic acid, HIF-1α, and IL-1β.
common up-regulated metabolites, which are cytidine, pyroglutamic
acid, succinic acid, and uracil (Fig. 5A-C).
Interestingly, the raised of intracellular succinic acid induced the 3.7. CRVP induces up-regulated expression of HIF-1α and down-
release of IL-1β [28]. We noted that stimulation of macrophages by regulated expression of PHD2 in macrophages
CRVP was accompanied by elevation of succinic acid and increased
levels of IL-1β transcription and translation. To sum up, we proposed the Previous studies have found that the expression of HIF-1α is regu-
following scientific hypothesis: Are the immunomodulatory effects of lated by many key genes, such as pyruvate kinase M2 (PKM2) and PHD2
CRVP on macrophage achieved by regulating the expression of IL-1 β [29,30]. By determining the expression of HIF-1α, PKM2, and PHD2 in
influenced by succinic acid? (Fig. 5D). macrophages, we verified that HIF-1α has high expression level and
PHD2 has low expression level after stimulating with CRVP, while the
result of PKM2 expression conflicting in RAW264.7 and THP-1 (Fig. 7A-
3.6. Succinic acid is related to the up-expression of IL-1β and HIF-1α in F). Thus, we further investigated the expression and link of HIF-1α and
CRVP-stimulated macrophages PHD2 at the protein level.
Western blot was used to better understand how CRVP affects the
To further confirm that macrophage stimulation by CRVP induced protein translation levels of HIF-1α and PHD2. We found that CRVP in
the accumulation of succinic acid, LC-MS was used for detecting the RAW264.7 significantly induced HIF-1α upregulation in high concen-
intracellular levels of succinic acid. Both in macrophage RAW264.7 and tration (20 μg/mL) and PHD2 downregulation compared with the CON
THP-1, succinic acid in the CRVP group was markedly elevated group (Fig. 7G and H). In THP-1, medium concentration CRVP (40 μg/
compared to the CON group (Fig. 6A and B), which was consistent with mL) led to an increase of HIF-1α, while there were inhibitory effects at
the result of metabolomics. high concentration (Fig. 7I). Likewise, the decrease of PHD2 happened
Thereafter, the succinic acid-rich culture experiment was designed to in THP-1 after treatment with CRVP and most significantly at 40 μg/mL
confirm whether succinic acid is a key metabolite in the immunomod- (Fig. 7J). Together, the results of western blot supported the results of
ulatory effect of CRVP. Succinic acid is an inflammatory signal, which RT-qPCR to some extent, and our results highlighted that the expression

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Fig. 3. CRVP increases oxidative stress in macrophages. NO release in CRVP-stimulated macrophage RAW264.7 (A), expression of NOS2 mRNA in CRVP-stimulated
macrophages THP-1 (B), and ROS levels in CRVP-stimulated macrophage RAW264.7 (C) and THP-1 (D), *p < 0.05, **p < 0.01, ***p < 0.001.

of HIF-1α and PHD2 in macrophages was regulated by CRVP, and there 4. Discussions
is a negative correlation between their expression.
In recent years, polysaccharides have been favored and applied by
3.8. HIF-1α inhibitor reverses CRVP-induced upregulation of HIF-1a and biochemical, pharmaceutical, food, and other industries due to their
IL-1β expression in macrophages multiple biological activities, high safety, and few or no side effects [16].
CRVP is a natural alga polysaccharide with rich sulfate moieties and
Next, we sought to confirm the underlying mechanism by which mannose [17]. Structural characteristics are the foundation of biological
PHD2 is inhibited, HIF-1α and IL-1β are activated in CRVP-stimulated activity in polysaccharides, and polysaccharides containing sulphate
macrophages and the HIF-1α inhibitor rescue experiment was adop- groups on sugar units showed great potential for immunological activity
ted. Before conducting the inhibitor rescue experiment, pre-treatment of [31]. Moreover, it has been reported that polysaccharides with high
RAW264.7 and THP-1 macrophages with a gradient concentration of mannose content can promote the production of NO, IL-6, and TNF-α,
LW6 (HIF-1α inhibitor) was carried out to verify the safety of LW6 which was attributed to the structures of mannan can bind with
application. Finally, LW6 with 10 μM was used as an effective safe mannose receptors on macrophage membranes to activate the immune
concentration for subsequent research (Fig. S5). In our results, treated system [32]. This led to an in-depth evaluation of the immunomodula-
with LW6 did not cause obvious changes compared to untreated in tory activity of CRVP.
RAW264.7. The gene expression of HIF-1α and IL-1β in the CRVP group Macrophages are an important component of the immune system,
could be reversed by administrating with LW6 and did not work in PHD2 originating from monocytes and playing a key role in innate and adap-
(Fig. 8A-C). Furthermore, similar to the gene expression, the protein tive immunity [33]. It is well known that RAW264.7 and THP-1 are
levels of HIF-1α and PHD2 analyzed with western blot showed the same commonly used cell lines for studying macrophage immunomodulatory
trends in RAW264.7(Fig. 8G-H). As for THP-1, inhibition of HIF-1α with activity, which can help support the validation of the biological activity
LW6 induced downregulation of genes HIF-1α, PHD2, and IL-1β in the of food compounds and the pharmacological activity of drugs in vivo
LW6 group, and the trend of changes in the CRVP group before and after [34,35]. In this study, both the RAW264.7 cell line and the THP-1 cell
the administration of LW6 was consistent with RAW264.7 (Fig. 8D-F and line were selected as research models to enable a broader evaluation of
I). However, LW6 enhanced the downregulation of PHD2 induced by the immunomodulatory activity of CRVP as well as to clarify the mo-
CRVP stimulation at the protein level, which was different from RT- lecular mechanisms by which it exerts its immunomodulatory activity in
qPCR (Fig. 8J). Generally speaking, the expression of HIF-1α and IL-1β macrophages. CCK-8 assay was adopted to determine the safe and
could be rescued by HIF-1α inhibitor. effective application concentration of CRVP and the result showed that
CRVP promoted macrophage proliferation when concentration at

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Fig. 4. The result of metabolomics. The volcano plot of DAMs in macrophage RAW264.7 (A) and THP-1 (B). The clustering heat map of significant DAMs in
macrophage RAW264.7 (C) and THP-1 (D). Distribution of KEGG pathways (p < 0.05) types in macrophage RAW264.7 (E) and THP-1 (F).

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Fig. 5. Succinic acid possibly contributes to IL-1β elevation in CRVP-stimulated macrophages. The Venn of joint metabolomics analysis (A). The content of co-DAMs
in macrophage RAW264.7 (B) and THP-1 (C). The immunomodulatory effects of CRVP on macrophage by regulating the level of intracellular succinate to affect the
expression of IL-1β (D).

2.5–20 μg/mL in RAW264.7 and 10-80 μg/mL in THP-1. It should be concentrations were selected for RAW264.7(5, 10, and 20 μg/mL) and
noted that CRVP is no longer promoted and may even inhibit macro- THP-1(20, 40, and 80 μg/mL) in following research.
phage proliferation at concentrations >40 μg/mL in THP-1. From this, As one of the most important indicators of immune regulation, cy-
we know that the effective concentration of CRVP varies for different tokines are essential for the maintenance of immune system homeosta-
cell lines, which confirms that RAW264.7 and THP-1 have different sis, and dysregulation of cytokine production can lead to an imbalance
tolerances and responses to the stimulus [26]. Thus, suitable in immune homeostasis and even result in the disease [21]. Both

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Fig. 6. Succinic acid is related to the up-expression of IL-1β and HIF-1α in CRVP-stimulated macrophages. The result of LC-MS in macrophages RAW264.7 (A) and
THP-1 (B). The gene expression of RAW264.7 (C and E) and THP-1 (D and F) in succinate-rich culture experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

macrophage RAW264.7 and THP-1 are known to have the ability to coincidentally. The result of the joint analysis showed that 7 DAMs were
cytokines secreted, and our study demonstrated that CRVP up-regulated co-enriched in non-targeted metabolomics of RAW264.7 and THP-1, and
the transcript levels of macrophage cytokines IL-6, IL-1β, and TNF-α, and 4 DAMs were co-upregulated, including cytidine, pyroglutamic acid,
promoted the secretion of the corresponding cytokines. Above, suggests succinic acid, and uracil. Among them, the expression of succinic acid
that CRVP can play an immunomodulatory role by modulating macro- treated with CRVP was >5 times higher than untreated group. Succinic
phage cytokine release. acid is an intermediate in the Krebs cycle, playing a crucial role in ATP
Redox-related mechanisms have been reported to regulate the production in mitochondria. And recent studies have shown that suc-
metabolic reprogramming of immune cells, and the function of indi- cinic acid is not only a metabolite, but also a class of molecules with
vidual immune cells is under redox control [23]. Intracellular ROS levels inflammatory signal transduction capabilities [10]. Some studies sug-
in macrophages affect STAT-1, MAPK, and NF-κB activity and lead to an gest that the accumulation of succinic acid in immune cells leads to
overall increase in inflammatory signaling [36]. In addition, ROS levels stabilization of HIF-1α or signaling through its receptors, which in turn
affect the assembly of NADPH oxidase subunits and regulate the for- enhances IL-1β production during inflammation [28]. In the present
mation of corrosive RNS substances (e.g. peroxynitrite), thereby influ- study, macrophages treated with CRVP induced the increase of succinic
encing H2O2-mediated intracellular signaling and macromolecular acid in vitro. It is also worth noting that the expression of IL-1β was been
damage [37]. NO is catalytically synthesized by inducible nitric oxide observed that significantly promoted by CRVP in previous studies.
synthase (iNOS), which is both a common indicator of the level of Herein, a scientific conjecture has been proposed: does CRVP achieve
oxidative stress within immune cells and an important immune effector immunomodulatory effects by inducing intracellular succinate accu-
molecule involved in immune regulation [23,38]. It is observed that mulation to affect the expression of IL-1β?
CRVP significantly induced NO production in RAW264.7 while causing Although the preliminary conjecture about the immunomodulatory
an increase in intracellular ROS levels in macrophages, which indicated mechanism of CRVP was put forward, there are still some questions that
that CRVP can play an immunomodulatory role by modulating the level urgently need to be further verified, for example, is the accumulation of
of oxidative stress in macrophages. succinic acid in macrophages induced by CRVP? What pathway does the
Since the “Warburg effect” was introduced at the beginning of the upregulation of IL-1β mediate by succinic acid? Thus, LC-MS was used to
20th century, biologists began to focus on metabolism research and now quantify succinic acid levels in macrophages in vitro after stimulating
scientists are finding that cellular metabolism plays a vital role in with CRVP, and the results reconfirmed that CRVP caused an increase in
advocating immune cell maintenance and development [39,40]. succinic acid content in macrophages compared to the control group.
Inspired by this, we attempted to elucidate the molecular mechanisms of Subsequently, succinic acid-enriched culture experiments showed that
CRVP immunomodulatory in macrophages from the perspective of different outcomes were presented in macrophages RAW264.7 and THP-
immunometabolism, which will further deepen the understanding of its 1 after treatment with succinic acid. For THP-1, treating with 10 μM
immunomodulatory activity. It is mentionable that the pathway type of succinic acid induced the upregulation of IL-1β and HIF-1α; for
“metabolism” was highlighted in non-targeted metabolomics both of RAW264.7, treating with 10 μM succinic acid could not significantly up-
RAW264.7 and THP-1, which supported the direction of our study regulate the transcript level of IL-1β and HIF-1α, which we inferred that

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J. Zeng et al. International Journal of Biological Macromolecules 277 (2024) 134450

Fig. 7. CRVP induces up-regulated expression of HIF-1α and down-regulated expression of PHD2 in macrophages. Gene expression in CRVP-stimulated macrophages
RAW264.7 (A-C) and THP-1 (D-F). The protein levels in CRVP-stimulated macrophages RAW264.7 (G and H) and THP-1 (I and J). *p < 0.05, **p < 0.01, ***p
< 0.001.

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J. Zeng et al. International Journal of Biological Macromolecules 277 (2024) 134450

Fig. 8. HIF-1α inhibitor reverses CRVP-induced upregulation of HIF-1α and IL-1β expression in macrophages. The gene expression of RAW264.7 (A-C) and THP-1 (D-
F) in HIF-1α inhibitor rescue experiment. The protein levels of RAW264.7 (G and H) and THP-1 (I and J) in the HIF-1α inhibitor rescue experiment. *p < 0.05, **p <
0.01, ***p < 0.001.

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J. Zeng et al. International Journal of Biological Macromolecules 277 (2024) 134450

macrophages initiate a protective mechanism to prevent homeostatic up-regulated in THP-1. Although the RT-qPCR results of PKM2 cannot
disturbances caused by excessive uptake of exogenous succinic acid directly reflect the expression levels of the two kinds of PKM2 configu-
[41]. What caught our attention was the association between the rations, it indicates whether CRVP can affect the expression of PKM2 to
expression levels of IL-1β and HIF-1α. Additionally, both the CRVP some extent. The elevated transcription level of PKM2 in THP-1 suggests
group and the CRVP+SA group elevated the expression of them. We that PKM2 may be involved in the immune regulation process of CRVP
supposed that HIF-1α is a key target that mediates between the accu- on THP-1, but this part of the work needs further investigation. Hence,
mulation of succinic acid and the production of the pro-inflammatory the expression of HIF-1α and PHD2 was measured at the protein level,
cytokine IL-1β. the results showed that the protein levels of PHD2 were decreased in
Glycolysis will be an important metabolic pathway in activated pro- RAW264.7 and THP-1 after being treated with CRVP. What’s more,
inflammatory macrophages, and one of the key targets of this process is RAW264.7 at 20 μg/mL and THP-1 at 40 μg/mL showed the most sig-
HIF-1α [42]. Hypoxia-inducible factor is an important regulator nificant upregulation effect of HIF-1α. The transcription and translation
involved in oxygen-dependent gene expression and consists of an levels of HIF-1in RAW264.7 were relatively consistent. However, there
oxygen-sensitive α-subunit (HIF-1α or HIF-2α) and a constitutively is a contradiction between the gene expression and the protein level of
expressed β-subunit [43]. PHD2, as an enzyme involved in HIF modifi- HIF-1α in THP-1, as the transcription level is still increasing while the
cation, and degradation can help stabilize the expression of HIF-1α [28]. translation level tends to remain unchanged or decrease. At the same
Meanwhile, PKM2 was found to play a crucial role in stabilizing and time, there are differences in the translation and transcription levels of
regulating HIF-1α, dimer PKM2 can form complexes with HIF-1α, PHD2. The above may be closely related to translation efficiency, pro-
inducing the production of IL-1β and the function of tetramer PKM2 is tein turnover, and post-translational modifications [44]. Substantially,
opposite to that of dimer PKM2 [29]. By determining gene expression CRVP promoted HIF-1α expression and repressed PHD2 expression.
with RT-qPCR, we found that HIF-1α was up-regulated and PHD2 was Importantly, the inhibitor rescue experiment was designed to further
down-regulated by CRVP, but PKM2 had no change in RAW264.7 and confirm whether HIF-1α is an important target for the

Fig. 9. The immunomodulatory activity of CRVP in macrophage via succinate/PHD2/HIF-1α/IL-1β pathway (red arrows indicate increasing levels, green arrows
indicate decreasing levels).

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J. Zeng et al. International Journal of Biological Macromolecules 277 (2024) 134450

immunomodulatory activity of CRVP in macrophages. In our study, the Data availability

up-regulated of HIF-1α and IL-1β treated with CRVP could be rescued by
LW6, the same as HIF-1α at the protein level. As for PHD2, previous I have shared the link to my data at the Supplementary File.
study proved that there is a correlation between HIF-1α and PHD2, and
the results showed that LW6 was unable to reverse the down-regulated Appendix A. Supplementary data
of PHD2 induced by CRVP, which also suggested PHD2 as an upstream
target affecting HIF-1α, and corroborates the existing view that inhibi- Supplementary data to this article can be found online at https://doi.
tion of PHD2 stabilizes the expression of HIF-1α [28]. Generally, the up- org/10.1016/j.ijbiomac.2024.134450.
regulated of succinic acid in macrophages to response to CRVP-
stimulation, which in turn destabilized PHD2 to maintain HIF-1α References
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