Invest Clin 66(2): 205 - 216, 2025 https://doi.org/10.54817/IC.v66n2a07
Corresponding author: Li Zhang. The First Clinical Medical College. Nanjing University of Chinese Medicine.
No. 138, Xianlin Road, Qixia District. Nanjing City 210023, Jiangsu Province, China. Tel: +86 13952009414.
Email: LiiZZhang@outlook.com
Lycopene regulates the formation of calcium
oxalate kidney stones by modulating
reactive oxygen species(ROS) and NF-κB
pathways.
Liangwen Ye, Yuhang Tang, Zhijie Zhang, Xiangyi Hou, Wei Xu,
Xianghui Suo and Li Zhang
The First Clinical Medical College, Nanjing University of Chinese Medicine,
Nanjing City, Jiangsu province, China.
Keywords: lycopene; renal tubular epithelial cells; oxalic acid; reactive oxygen species;
apoptosis.
Abstract. This study aims to determine whether lycopene can reduce oxi-
dative stress and inflammatory damage in HK-2 cell cultures induced by cal-
cium oxalate crystallization through the modulation of reactive oxygen species
(ROS) and the NF-κB signalling pathway. Cell cultures were divided into four
groups: The control group, the Model group (COM + oxalic acid), and two Lyco-
pene intervention groups (COM + oxalic acid + 5/10 μmol/L lycopene). After
24 hours of culture, viability, LDH, oxidative and anti-oxidative parameters, mi-
tochondrial membrane potential, MCP-1, IL-6, apoptosis and related proteins,
and activation and expression of NF-κB were determined by adequate methods.
When compared to the control group, the model group exhibited decreased cell
activity (p<0.001) and GSH and SOD antioxidant capacity (p<0.05), along-
side a significant rise in LDH, MDA, and the release of inflammatory mediators
MCP-1 and IL-6 (p<0.05). The levels of protein expression for NF-κB, OPN, Bax,
Cyt C, and active Caspase-3 were increased (p<0.05), whereas Bcl-2 protein
expression significantly diminished (p<0.05). The mitochondrial membrane
potential decreased. Lycopene intervention reduced the damage to HK-2 cells
(p<0.05), accompanied by decreased levels of LDH, MDA, and inflammatory
factors MCP-1 and IL-6 (p<0.05), and increased GSH and SOD antioxidant
capacity (p<0.05). The mitochondrial membrane potential was observed to in-
crease. No significant changes were observed in the expression of NF-κB. The
expressions of OPN, Bax, Cyt C, and Caspase-3 decreased (p<0.05), whereas
the level of Bcl-2 protein expression increased. In conclusion, lycopene decreas-
es cellular damage by inhibiting lipid peroxidation induced by calcium oxa-
late crystals and oxalate, enhancing intracellular antioxidant enzyme activity,
modulating ROS and NF-κB inflammatory pathways, improving mitochondrial
integrity, and exerting anti-inflammatory effects through the inhibition of the
mitochondrial-mediated Bax/Caspase-3 signalling pathway.
206 Ye et al.
Investigación Clínica 66(2): 2025
El licopeno regula la formación de cálculos renales de oxalato
cálcico modulando las vías de especies reactivas de oxígeno
(ROS) y NF-κB.
Invest Clin 2025; 66 (2): 205 – 216
Palabras clave: licopeno; células epiteliales tubulares renales; ácido oxálico; especies
reactivas de oxígeno; apoptosis.
Resumen. Este estudio tiene como objetivo determinar si el licopeno puede
reducir el estrés oxidativo y el daño inflamatorio inducidos por la cristalización
de oxalato de calcio en cultivos de células HK-2 a través de la modulación de es-
pecies reactivas de oxígeno (ROS) y las vías de señalización de NF-κB. Los cultivos
celulares se dividieron en cuatro grupos: grupo control, grupo modelo (COM +
ácido oxálico) y dos grupos de intervención con licopeno (COM + ácido oxálico
+ 5/10 μmol/L de licopeno). Después de 24 horas de cultivo, se determinaron
la viabilidad, la LDH, los parámetros oxidativos y antioxidantes, el potencial de
membrana mitocondrial, MCP-1, IL-6, la apoptosis y proteínas relacionadas, y la
activación y expresión de NF-κB mediante métodos adecuados. En comparación
con el grupo control, el grupo modelo mostró una actividad celular (p<0,001)
y una capacidad antioxidante de GSH y SOD (p<0,05) disminuidas, junto con
aumento significativo de LDH, MDA y la liberación de mediadores inflamatorios
MCP-1 e IL-6 (p<0,05). Los niveles de expresión de proteínas para NF-κB, OPN,
Bax, Cyt C y Caspasa-3 activa aumentaron (p<0,05), mientras que la expresión
de la proteína Bcl-2 disminuyó significativamente (p<0,05). El potencial de
membrana mitocondrial disminuyó. La intervención con licopeno redujo el daño
celular (p<0,05), acompañada de una disminución de los niveles de LDH, MDA y
los factores inflamatorios MCP-1 e IL-6 (p<0,05), y un aumento de la capacidad
antioxidante de GSH y SOD (p<0,05). Se observó un aumento del potencial de
membrana mitocondrial. No se observaron cambios significativos en la expresión
de NF-κB. La expresión de OPN, Bax, Cyt C y Caspasa-3 disminuyó (p<0,05),
mientras que la expresión de la proteína Bcl-2 aumentó. En conclusión, el lico-
peno disminuyó el daño celular al inhibir la peroxidación lipídica inducida por
cristales de oxalato de calcio y oxalato, potenciar la actividad enzimática antioxi-
dante intracelular, modular las vías inflamatorias de ROS y NF-κB, mejorar la in-
tegridad mitocondrial y ejercer efectos antiinflamatorios mediante la inhibición
de la vía de señalización Bax/Caspasa-3 mediada por mitocondrias.
Received: 25-03-2025 Accepted: 27-04-2025
INTRODUCTION
Kidney stones constitute a common is-
sue encountered in the urology field. Their
widespread occurrence, high rates of recur-
rence, and the financial burden of treatment
have significant implications for individu-
als and society 1. One key factor contribut-
ing to kidney stone formation is the harm
inflicted on renal tubular epithelial cells
due to increased oxalate levels, with hyper-
oxaluria identified as a significant risk fac-
Lycopene and kidney stones: ROS-NF-κB link 207
Vol. 66(2): 207 - 216, 2025
Macklin Biochemical Technology Co., Ltd.,
product number O871905), DMEMF-12 (1:1)
basic medium (Gibco, USA, product number
C11330500BT), Cell Counting Kit-8 (Biosharp,
product number BS350B), Reduced gluta-
thione (GSH) assay kit, Lactate dehydroge-
nase (LDH) assay kit (Nanjing Jiancheng
Bioengineering Institute Co., Ltd., product
numbers A006-2-1 and A020-2-2), Malondial-
dehyde (MDA) Colorimetric Assay Kit, Total
Superoxide Dismutase (T-SOD) Activity Assay
Kit (Wuhan Elabscience Biotechnology Co.,
Ltd., product numbers E-BC-K028-M and E-
BC-K020-M), In this study, we employed the
human IL-6 ELISA kit and the human MCP-1
ELISA kit (Quanzhou Ruixin Biotechnology
Co., Ltd., product numbers RX106126H and
RX106032H), Reactive oxygen species(ROS)
detection kit(Shanghai beyotimeBiotechnol-
ogy Co., Ltd., product numbers S0033S). Ad-
ditionally, rabbit-derived antibodies include
NF-κB p65, Osteopontin (OPN), Bax, Bcl-2,
cytochrome C (Cyt C), and active-Caspase3.
Secondary antibodies include an anti-mouse
antibody from Shanghai Beyotime Biotech-
nology Co., Ltd. (product numbers: AF5243,
AF7662, A0216) and a secondary rabbit an-
tibody from Proteintech Group, Inc. (batch
numbers: 50599-2-Ig, 26593-1-AP, SA00001-
2). Furthermore, a mouse-derived GAPDH an-
tibody from BOSTER is identified by product
numbers: PB9334, BM3937, and BM3876.
Instrumentation
In this research, the equipment used
included the Series II Water Jacket CO2 cell
culture incubator, the Infinite M1000 Pro full-
wavelength microplate reader (Tecan, Switzer-
land), the Axio Vert A1 inverted fluorescence
microscope (Zeiss, Germany), the Mini-Pro-
tean 3 Dodeca electrophoresis system, the
ChemiDoc XPS+ all-in-one gel imaging system
(Bio-Rad Company, USA), and the MoFlo XDP
ultra-fast flow cytometer (BD Company, USA).
Method
LYC was dissolved in DMSO, and a blank
culture medium was subsequently introduced
tor for developing urinary stones 2. Exposure
to elevated concentrations of oxalic acid
over extended periods can trigger oxidative
stress in these cells, leading to an overpro-
duction of reactive oxygen species. This pro-
cess may cause cellular harm, such as cell
degeneration, apoptosis, and the exposure
of the basement membrane of renal tubular
epithelial cells 3, potentially worsening sub-
sequent injuries. Following this, a series of
cellular lipid peroxidation and inflammatory
responses may occur 4; as a result, antioxi-
dants and anti-inflammatory medications
are commonly employed to avert renal injury
and the formation of kidney stones.
Lycopene (LYC), a vital carotenoid
that falls under the classification of iso-
prenoid compounds, demonstrates proper-
ties such as anti-inflammatory, antioxidant,
free radical scavenging, and immune modu-
lation 5, 6. Research indicates that lycopene
may aid in relieving chronic prostatitis/
chronic pelvic pain syndrome through its
ability to diminish inflammation and oxida-
tive stress by engaging the NF-κB, Nrf2, and
MAPKs signalling pathways 7. Nonetheless,
no prior investigations have directly exam-
ined its protective effects against kidney
damage caused by calcium oxalate stones.
This study intends to explore the role and
associated molecular mechanisms of LYC
in the damage inflicted on renal tubular
epithelial cells by oxalic acid and calcium
oxalate crystals in vitro, thereby providing
a theoretical foundation for utilizing anti-
inflammatory and antioxidant agents, such
as LYC, in the prevention and management
of kidney stone disorders.
MATERIAL AND METHODS
Cells. HK-2 cells (purchased from BOS-
TER, catalogue number CX0044) were pas-
sed to the ninth passage.
Drugs and Reagents. Lycopene (Shanghai
Yuanye Biotechnology Co., Ltd., product num-
ber B20378, purity ≥90%), oxalic (Shanghai
208 Ye et al.
Investigación Clínica 66(2): 2025
to formulate a storage solution with a con-
centration of 1000 μmol/L. The solution was
passed through a microporous filter with a
pore size of 0.22 μm and kept in a refrigera-
tor at 4°C. Before beginning the experiment,
the prepared LYC solution was administered
to cultured cells in increasing concentrations
(5, 10, 20, 40, 80, 100, 200, 500 μmol/L) to
identify LYC’s effective concentration and
toxicity range. In a 96-well plate populated
with HK-2 cells (1×104 cells/well), the ef-
fects of different LYC concentrations on HK-2
cytotoxicity were evaluated using the CCK-8
kit, with assessments made 24 hours after
administration (refer to Table 1). Ultimately,
lycopene concentrations of 5 μM and 10 μM
were chosen for further experiments.
“A” Experimental grouping and inter-
vention
The experimental groups were defined
as follows: 1) Control group: cultured in
basal medium for 24 hours; 2) Model group:
cultured in basal medium containing oxalic
(2 mmol/L) and COM (100 μg/mL) for 24
hours; 3) LYC I (5 μmol/L) group: treated
with 5 μmol/L (LYC) in addition to the mod-
el group; 4) LYC II (10 μmol/L) group: treat-
ed with 10 μmol/L LYC in addition to the
model group. These groups were utilized for
subsequent experiments, including cell vi-
ability assessments, antioxidant capacity, in-
flammatory factors, reactive oxygen species,
and Western blot analysis. Before the experi-
ments, the original medium in the culture
wells was removed, and serum-free medium
was added to minimize the influence of pro-
teins present in fetal bovine serum (FBS) on
the experimental outcomes.
“B” CCK-8 assay to detect cell activity
HK-2 cells were plated in a 96-well plate at
a density of 1 × 10^4 cells per well. After over
18 hours for complete attachment, the cells
were allocated into groups for experimental
interventions as specified in Section “A”. Once
the interventions were completed, the original
culture medium was discarded and substituted
with serum-free medium in every well. Subse-
quently, 100 μL of newly prepared culture me-
dium and 10 μL of CCK-8 reagent were added,
and the plate was incubated at 37°C for three
hours. The absorbance (Ab) at 450 nm was re-
corded using a microplate reader. Each condi-
tion was evaluated in parallel within six repli-
cate wells, and the experiment was conducted
three times. The average value of Ab was com-
puted, and cell activity was evaluated using the
formula: Ab (experimental group) / Ab (con-
trol group) × 100%.
“C” Measurement of indicators related
to antioxidant capacity
Cells were plated in a 6-well culture plate
at a density of 2 x 105 cells per well until they
adhered properly. The experimental groups
aligned with those described previously (Ex-
perimental grouping). After a 24-hour incu-
Table 1. Effect of different concentrations of Lycopene on HK-2 cell activity.
Groups Concentration /μmol/L Relative cellular activity / %
Control group
Lycopene group
0
5
10
20
40
80
100
200
500
100 ± 15.92
98.26 ± 1.09
94.75 ± 0.84
88.13 ± 2.12
86.92 ± 2.75
85.02 ± 2.47
79.82 ± 2.34
69.27 ± 4.76
31.39 ± 2.42
Data is expressed as ± sd, n=3.
Lycopene and kidney stones: ROS-NF-κB link 209
Vol. 66(2): 207 - 216, 2025
bation, cells from each group were collected,
and the protein concentration was measured
in centrifuge tubes. The instructions of the
kit were followed to operate. Finally, the
contents of lactate dehydrogenase(LDH),
malondialdehyde (MDA), glutathione(GSH),
and total superoxide dismutase(T-SOD) were
measured in the cells using a Microplate
reader at wavelengths of 450, 532, 405, and
450 nm and an ELISA kit was used for detec-
tion of IL-6, MCP-1 secretion.
“D” Cells were plated in a 6-well culture
plate at a density of 2 x 105 cells per well un-
til they adhered properly. The experimental
groups aligned with those described in section
“A”. After a 24-hour incubation, the superna-
tant from each cell group was gathered into
centrifuge tubes. Next, 50 μL from each group
was transferred to the enzyme plate, following
the instructions provided with the kit. Essen-
tial procedures included preparing three dupli-
cate wells for every cell group, with the experi-
ment conducted three times. The absorbance
(Ab) measurement was taken at a wavelength
of 450 nm to assess the levels of the inflamma-
tory cytokines IL-6 and MCP-1.
“E” Observation of cellular ROS
Cells were plated in a 6-well culture dish
at a density of 2 x 105 cells per well, adhering to
the group allocations outlined before (A). After
a 24-hour culture period, a ROS detection kit
was utilized to evaluate the levels of intracel-
lular ROS. Specifically, 10 μmol/L DCFH-DA,
which was diluted in serum-free culture me-
dium, was introduced in a dark environment.
One mL of this fluorescent probe was admin-
istered, and the cells were incubated for 20
minutes. After incubation, the cells underwent
three washes with 1 mL of serum-free culture
medium, after which images were taken using
an inverted fluorescence microscope.
“F” Western blot for protein expression
in HK-2 cells
Cells were planted in a 6-well plate ac-
cording to the method described in Section
“D”, then collected and denatured at high
temperature. Electrophoresis was performed
using the SDS gel system, followed by trans-
fer to a PVDF membrane. The membrane
was blocked with 5% skim milk at room tem-
perature for 2 hours. The primary antibodies
(NF-κB p65, OPN, Bax, Bcl-2, Cyt C, active-
Caspase3, and GAPDH) were added and in-
cubated at 4°C overnight. Subsequently, they
were incubated with a secondary antibody at
room temperature for 1.5 hours. The develop-
ing agent was added, and images were cap-
tured using an automatic gel imager. The Im-
ageJ software was used to measure the gray
value of each band and calculate the relative
expression of target proteins in each group.
Statistical methods
All data were analyzed statistically with
the use of GraphPad Prism 8.0.1 software. The
outcomes are represented as ± SD. A one-
way ANOVA was utilized to compare several
groups. A p-value lower than 0.05 was consid-
ered a statistically significant difference.
RESULTS
Comparison of cell activity and LDH
in each group
Following 24 hours of treatment in the
culture medium, the viability of cells in each
group was assessed. The findings indicated
a notable reduction in cell viability within
the model group when juxtaposed with the
control group (p<0.001), alongside a sig-
nificant elevation in LDH levels (p<0.001).
Conversely, cell viability in the LYC groups
(5 μM, 10 μM) exhibited a marked increase
compared to the model group (p<0.05),
while LDH levels showed a significant de-
crease (p<0.05) (Table 2).
Comparison of antioxidant and anti-
inflammatory capacities of HK-2 cells
The findings from the biochemical in-
dex assessments indicated a notable reduc-
tion in GSH levels within the model group
when contrasted with the control group
210 Ye et al.
Investigación Clínica 66(2): 2025
(p<0.05). Conversely, MDA levels were
found to have increased markedly (p<0.05).
MDA levels saw a significant decline due
to LYC treatment (p<0.001) (Table 3).
Furthermore, when examining the model
group, there was a notable surge in inflam-
matory cytokines IL-6 and MCP-1 compared
to the control group (p<0.05). LYC admin-
istration at doses of 5 μM and 10 μM dem-
onstrated an inhibitory effect on the secre-
tion of IL-6 and MCP-1 relative to the model
group (p<0.05) (Table 4).
Changes in intracellular ROS,
mitochondrial membrane potential and
apoptosis across different cell groups
In comparison to the control group, the
model group exhibited enhanced green fluo-
rescence and a reduced mitochondrial mem-
brane potential. Following intervention with
LYC (5 μM, 10 μM), the model group showed
an increase in red fluorescence, a decrease
in green fluorescence, and an improvement
in mitochondrial membrane potential. More-
over, the generation of ROS was increased
in the model group compared to the control
group. However, after LYC intervention (5
μM, 10 μM), the model group demonstrated
a decrease in ROS production (see Fig. 1 and
Fig. 2). PI and Hoechst staining indicated
that, in contrast to the control group, there
was a rise in apoptotic cells within the model
group, evidenced by intensified blue fluores-
cence. In comparison to the model group,
treatment with LYC (5 μM, 10 μM) resulted
in an improvement and a reduction in cell
apoptosis, as shown by diminished blue fluo-
rescence (Fig. 3).
Expression of inflammation and apoptosis-
related proteins
The levels of NF-κB p65 and OPN in
the model group were significantly ele-
vated (p<0.05) compared to the control
group, whereas LYC (5 μM, 10 μM) led to a
decrease relative to the model group (Fig.
4). The expression levels of Bax, CytC, and
active caspase3 were markedly increased
(p<0.05) in the model group when com-
pared to the control group, while Bcl-2
expression was significantly decreased
(p<0.05). The LYC (5 μM, 10 μM) treat-
ment group exhibited a downregulation
in Bax, CytC, and active caspase3 expres-
sion, along with an upregulation in Bcl-2
expression compared to the model group
(Table 5, Fig. 5).
DISCUSSION
The main goals in treating kidney stones
include removing the stones, protecting kid-
ney function, and tackling the root causes to
reduce the likelihood of recurrence 8. Thus,
it is vital to identify specific pharmacologi-
cal agents that target the condition’s etiol-
ogy for preventing and treating stones. The
formation of kidney stones is a complicated
process that entails the supersaturation of
factors contributing to urolithiasis, harm to
renal tubular epithelial cells, and the mecha-
nisms of crystal adhesion, aggregation, nucle-
ation, and growth 9. A significant contributor
to the development of kidney stones is oxalic
acid. Elevated concentrations of oxalic acid
may result in oxidative damage and initiate
an inflammatory reaction in renal tubular
epithelial cells 10.
Table 2. Comparison of cellular activity and intracellular lactate dehydrogenase content
in each group.
Items Control group Model group LYC (5μM) LYC (10μM)
Cellular activity (%)
LDH (U/g prot)
100
369.9±41.43
42±2.98a
906±79.97a
60.37±3.44b
483.7±70.8b
51.79±1.88b
588.8±33.56b
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group; as x, ± sd,
n=3. LDH: lactate dehydrogenase; LYC: Lycopene.
Lycopene and kidney stones: ROS-NF-κB link 211
Vol. 66(2): 207 - 216, 2025
An expanding array of studies has dem-
onstrated a relationship between inflamma-
tion, oxidative stress, and kidney stone for-
mation 4, 11. Hence, investigating effective
anti-inflammatory and antioxidant mecha-
nisms is crucial for alleviating kidney injury
associated with calcium oxalate stones.
Malondialdehyde (MDA) is the end
product generated from the peroxidation
of cellular lipids. The levels of MDA offer
valuable information regarding the degree
of lipid peroxidation in the body, thereby
acting as an indirect indicator of cellular
damage.
Although free radicals can inflict con-
siderable harm, human cells also harbor
substances that neutralize these free radi-
cals. Among these protective agents, super-
Table 3. Comparison of indicators related to intracellular antioxidant capacity in each group.
Items Control group Model group Lycopene
(5μM)
Lycopene
(10μM)
Glutathione
(GSH, umol/g protein)
Total superoxide dismutase
(T-S0D, U/mg protein)
Malondialdehyde
(MDA, nmol/mg protein)
327.4±29.98
13.54±0.70
2.166±0.3
111.8±11.71a
11.57±1.61
22.89±0.441a
212.1±21.1b
15.03±2.56
14.68±1.72b
201.2±22.19b
14.59±2.29
15.43±0.85b
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group. The above
data was analyzed using one-way ANOVA. ± sd, n=3.
Table 4. Effect of Lycopene on Interleukin-6 (IL-6) and Monocytechemotactic protein-1 (MCP-1)
released from oxalic acid/calcium oxalate-induced HK-2 cells.
Groups IL-6 (pg/mL) MCP-1(pg/mL)
Control group
Model group
Lycopene (5μM)
Lycopene (10μM)
4.49 ± 0.44
9.25 ± 0.46a
5.42 ± 1.49b
5.28 ± 1.51b
9.78 ± 1.38
29.38 ± 3.51a
7.71 ± 6.10b
11.84 ± 4.85b
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group. The above
data was analyzed using one-way ANOVA; ( ± sd, n=3).
Fig. 1. Effect of LYC on oxalic acid/calcium oxalate crystal-induced intracellular reactive oxygen species ROS
in HK-2 cells. A. control group; B. model group; C. LYC (5 μM); D. LYC (10 μM) group (Immunofluo-
rescence, x100).
212 Ye et al.
Investigación Clínica 66(2): 2025
oxide dismutase (SOD) stands out as a key
antioxidant enzyme that aids in reducing
the damage inflicted by oxygen-derived free
radicals 12. A reduction in SOD activity in-
dicates a lower ability of the organism to
combat free radical-induced damage, imply-
ing that the organism may be undergoing
oxidative stress. For example, continuous
exposure to elevated levels of oxalic acid
can promote the production of free radi-
cals, which initiate lipid peroxidation with-
in biological membranes.
Fig. 2. Effect of LYC on mitochondrial membrane potential induced by oxalic acid/calcium oxalate crystals
in HK-2 cells. A. control group; B. model group; C. LYC (5 μM); D. LYC (10 μM) group (Immunofluo-
rescence, x100).
Fig. 3. Effect of LYC on the apoptotic profile of HK-2 cells induced by oxalic acid/calcium oxalate crystals. A.
control group; B. model group; C. LYC (5 μM); D. LYC (10 μM) group. PI: propidium iodide (Immu-
nofluorescence, x100).
Lycopene and kidney stones: ROS-NF-κB link 213
Vol. 66(2): 207 - 216, 2025
This chain of events may result in chang-
es to the ultrastructural integrity of cell
membranes, enable cellular penetration, in-
flict harm on mitochondria and DNA, and ul-
timately lead to cell necrosis and apoptosis13.
These experimental findings reveal that the
oxidative damage to HK-2 cells induced by
oxalic acid/calcium oxalate is significant, as
evidenced by compromised cell proliferation,
decreased cell viability, heightened release of
lipid peroxidation byproducts like MDA and
LDH, along with lower levels of antioxidant
enzymes such as SOD and GSH. Following
treatment with LYC, we noted an enhance-
ment in cell viability, reduced oxidative in-
jury, and increased antioxidant activity.
Reactive oxygen species (ROS) are rec-
ognized as primary regulators of oxidative
stress and identified as significant contrib-
utors to the damaging effects of pathologi-
Table 5. Relative expression of proteins related to inflammation and mitochondrial damage
in cells of each group.
Items Control group Model group Lycopene (5μM) Lycopene(10μM)
P65
Osteopontin (OPN)
BAX
Bcl-2
Cytochrome C (Cyt C)
Active caspase3
0.708±0.077
0.546±0.70
0.427±0.062
1.13±0.301
0.539±0.066
0.355±0.155
1.014±0.053a
0.864±0.006a
1.109±0.205a
0.466±0.228a
2.242±0.428a
1.16±0.231a
0.807±0.163
0.746±0.260
0.734±0.221
0.536±0.261
0.930±0.264b
0.585±0.212b
0.782±0.229
0.642±0.127b
0.542±0.203b
0.544±0.245
0.775±0.490b
0.771±0.165
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group. The above
data was analyzed using one-way ANOVA.
Fig. 4. LYC ameliorates oxalic acid/calcium oxa-
late crystal-induced changes in inflam-
mation-associated protein levels in HK-2
cell injury. Fig. 5. Changes in the levels of mitochondrial da-
mage-related proteins in HK-2 cells.
cal stone formation 14. The role of the NF-κB
signaling pathway is critical in facilitating
intrarenal inflammation mediated by oxi-
dative stress 15. The significant production
of ROS caused by oxidative stress can addi-
tionally activate various signalling pathways
associated with inflammation, creating di-
verse inflammatory mediators and promot-
ing the buildup of inflammatory cells, thus
triggering and enhancing the inflammatory
response. Studies show that heightened ox-
alic acid levels can provoke the activation of
the NF-κB intracellular signalling pathway,
which leads to an increased expression of
inflammatory factors like OPN and MCP-1/
IL-6, ultimately resulting in the infiltra-
tion of inflammatory cells and causing in-
terstitial damage 4. Monocyte chemotactic
protein-1 is an essential inflammatory me-
diator contributing to the inflammatory
214 Ye et al.
Investigación Clínica 66(2): 2025
reactions linked to calcium oxalate kidney
stones. Under standard physiological con-
ditions, renal tissue cells only produce a
minimal amount of MCP-1. However, when
oxalic acid or calcium oxalate crystals pre-
cipitate in urine due to supersaturation,
these substances can damage and stimulate
these cells, leading to a substantial increase
in MCP-1 production, subsequently attract-
ing monocytes into the inflamed tissue 16.
Boonla et al. 17 compared MCP-1 and IL-6
mRNA expression levels in kidney tissues
adjacent to stones and those in normal kid-
neys. Their findings indicated more severe
tubular damage in the tissues surrounding
the stones and significantly reduced expres-
sion levels of MCP-1 and IL-6 compared to
normal kidney tissues. This observation im-
plies that MCP-1 and IL-6 may be involved
in advancing kidney stone disease.
Furthermore, during cellular damage,
various negatively charged molecules—in-
cluding osteopontin (OPN), hyaluronic acid
(HA), and CD44 18—are showcased on the
cell surface. These molecules can bind to
Ca2+ ions and attach to positively charged
calcium oxalate crystals. The adhering crys-
tals can trigger cellular production of free
radicals, further harming the renal epithe-
lium via lipid peroxidation, thus heighten-
ing the possibility of kidney stone forma-
tion. The study utilized ELISA experiments
to show that MCP-1 and IL-6 release were
elevated in the model group, while LYC was
found to have a protective effect. Western
blot analyses indicated that NF-κB and OPN
levels were upregulated in the model group
relative to the control group; however, their
expression was reduced after LYC treat-
ment. In addition, a combined analysis of re-
active oxygen species levels in cells revealed
that LYC could ameliorate the intrarenal
ROS levels triggered by oxalic acid/calcium
oxalate crystals in HK-2 cells, effectively in-
hibiting the NF-κB signalling pathway and
thereby diminishing the intrarenal inflam-
matory response.
The deposition of calcium oxalate crys-
tals may also cause damage to mitochondria
by increasing cellular ceramide levels. Mito-
chondrial abnormalities or oxidative stress
can trigger the initiation of cell apoptosis
programs. The family of Bcl-2 proteins is cru-
cial in governing mitochondrial permeability
to various proteins and the permeabilization
of the outer mitochondrial membrane, play-
ing an essential role in the intrinsic apoptosis
pathway. Bax and Bcl-2 are pro-apoptotic and
anti-apoptotic agents, respectively 19. While
Bcl-2 shields cells from mitochondrial in-
jury and suppresses apoptosis, Bax enhances
the permeability of the mitochondrial mem-
brane, facilitating the release of cytochrome
C (Cyt C) 20. This sequence of events leads to
an increase in hydrogen peroxide production,
a decrease in glutathione peptide levels, and
a drop in mitochondrial membrane potential,
coupled with the liberation of apoptotic fac-
tors into the cytosol, which ultimately acti-
vates caspase-3 and induces cell death 21. The
expression levels of proteins associated with
the mitochondrial pathway were evaluated in
this study.
Results indicate that treatment with ox-
alic acid/calcium oxalate crystals led to an
upregulation of Bax, Cyt C, and active-cas-
pase3, while a downregulation of Bcl-2 was
observed. Following the intervention with
LYC, improvements in cell apoptosis were
noted. These findings imply that LYC may
mitigate HK-2 cell apoptosis by inhibiting the
Bax/caspase3 signalling pathway.In conclu-
sion, LYC demonstrates a significant ability
to reduce oxidative stress and inflammatory
responses in HK-2 cells, enhances cellular
health, and may operate through the modula-
tion of the ROS/NF-κB inflammasome path-
way while also mitigating mitochondrial dam-
age by inhibiting the Bax/caspase3 signalling
pathway associated with mitochondria. This
research offers an initial insight into the po-
tential mechanisms by which LYC could aid
in the clinical prevention and treatment of
kidney stones, thereby opening new avenues
Lycopene and kidney stones: ROS-NF-κB link 215
Vol. 66(2): 207 - 216, 2025
and concepts for addressing calcium oxalate
kidney stones and the clinical utilization of
LYC and analogous medications. Nonethe-
less, as the investigation primarily focuses on
cellular models, it may not wholly replicate
the mechanisms involved in the human body,
indicating that the study has inherent limi-
tations, warranting further exploration and
validation.
ACKNOWLEDGEMENTS
Thanks for the support from the Special
Project for the Promotion of Department
Heads of Traditional Chinese Medicine Hos-
pitals in Jiangsu Province.
Funding
This study was supported by the Special
Project for the Promotion of Department
Heads of Traditional Chinese Medicine Hos-
pitals in Jiangsu Province(Y2021Z19).
Ethics approval and consent to participate
Not applicable, our study utilizes com-
mercially available cell lines, and the Nan-
jing University of Chinese Medicine does not
mandate ethics review for research conduct-
ed with such cell lines. So, there are no ethi-
cal issues or conflicts of interest.
Availability of data and materials
The datasets used and/or analyzed dur-
ing the current study are available from the
corresponding author on reasonable request.
Conflict of interests
All authors declare no conflict of interest.
Number ORCID of authors
• Liangwen Ye (LY):
0009-0003-9780-4688
• Yuhang Tang (YT):
0009-0009-6263-0570
• Zhijie Zhang (ZZ):
0009-0006-8535-0748
• Xiangyi Hou (XH):
0009-0004-5185-6965
• Wei Xu. (WX):
0009-0004-0948-5822
• Xianghui Suo (XS):
0009-0008-1688-0694
• Li Zhang (LZ):
0009-0002-2514-6690
Authors’ Contributions
LY and YT designed the study; data ac-
quisition, analysis, and interpretation were
conducted by ZZ and XH; the manuscript
underwent revision by LY, WX, and XS; LZ
also played a role in the article’s revision.
Each author contributed to the paper and
approved the final version submitted.
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