Invest Clin 67(2): 241 - 259, 2026 https://doi.org/10.54817/IC.v67n2a07

Corresponding author: Yao Lu. Department of Thoracic Surgery, The Fourth Affiliated Hospital of China Medical

University, Shenyang City, Liaoning Province, China. Email: luyao52611@hotmail.com

Orientin attenuates pulmonary fibrosis

via TGF-β1/suppressor of mother against

decapentaplegic 3 (Smad3) pathway.

Yijin Liu1 and Yao Lu2

1Department of Pharmacy, Liaoning University of Traditional Chinese Medicine Xinglin

College, Shenyang, Liaoning Province, China.

2Department of Thoracic Surgery, The Fourth Affiliated Hospital of China Medical

University, Shenyang City, Liaoning Province, China.

Keywords: Orientin; Pulmonary fibrosis; Fibroblasts; TGF-β1/Smad3 pathway.

Abstract. Orientin, a natural flavonoid found in many medicinal plants, can

improve lung injury through anti-inflammatory and antioxidant effects, but its

role in pulmonary fibrosis (PF) remains unstudied. Human Fetal Lung 1 (HFL1)

cells were stimulated with transforming growth factor-β1 (TGF-β1), and a single

intratracheal bleomycin instillation in mice was used to establish a PF mouse

model. Orientin, the TGF-β1/suppressor of mother against decapentaplegic 3

(Smad3) pathway agonist SRI-011381, and the inhibitor SB431542 were used

for intervention. The proliferation and migration were evaluated using the Cell

Counting Kit-8 (CCK-8), Edu staining (evaluated proliferative activity) and a

scratch-healing assay. Fibers in HFL1 cells were detected by Sirius red staining.

Inflammation and fibrosis in lung tissue were assessed by pathological staining

and enzyme-linked immunosorbent assay (ELISA). PF and TGF-β1/Smad3 path-

way protein expressions were evaluated by Western blot. Orientin significantly

reduced TGF-β1, p-Smad3, alpha-smooth muscle actin (α-SMA), Collagen I, and

matrix metallopeptidase (MMP)-2 levels. After Orientin treatment, the Edu pos-

itive cells, cell proliferation and migration were significantly reduced, and the

number of red-stained collagen fibers was significantly reduced. After Orientin

treatment, alveolar cavity collapse, inflammatory cell infiltration, and collagen

fiber hyperplasia of mice were alleviated, and the contents of Hydroxyproline

(HYP) and inflammatory factors in the alveolar lavage fluid were significantly

reduced. SRI-011381 attenuated the effect of Orientin on the intervention, and

inflammation and fibrosis levels were markedly increased. SB431542 enhanced

the intervention effect of Orientin. Orientin inhibited TGF-β1/Smad3 signal-

ing, inhibited fibroblast-to-myofibroblast transition (FMT) and extracellular

matrix (ECM) production, and alleviated inflammatory and fibrotic damage.

242 Liu and Lu

Investigación Clínica 67(2): 2026

La orientina reduce la fibrosis pulmonar mediante la vía

de señalización TGF-β1/Smad3.

Invest Clin 2026; 67 (2): 241 – 259

Palabras clave: Orientin; Fibrosis pulmonar; Fibroblastos; Vía de TGF-β1/Smad3.

Resumen. La orientina, un flavonoide natural presente en una variedad de

plantas medicinales, reduce el daño pulmonar gracias a sus efectos antiinfla-

matorios y antioxidantes. Sin embargo, no se han reportado estudios sobre su

efecto en la fibrosis pulmonar (PF). Las células Human Fetal Lung 1 (HFL1) se

estimularon con el factor de crecimiento transformante β1 (TGF-β1) y se admi-

nistró una dosis única de bleomicina por vía intratraqueal para establecer un

modelo de PF en ratones. La intervención se realizó con orientina, el agonista

SRI-011381 de la vía supresora de madre contra el decapentapléjico 3, y el in-

hibidor SB431542. La proliferación y migración de células HFL1 se evaluaron

mediante el Kit-8 (CCK-8) para el conteo de células, la tinción de Edu (para eva-

luar la actividad proliferativa) y mediante el ensayo de cicatrización de heridas.

Las fibras en las células HFL1 se detectaron mediante tinción con Sirius Red.

La inflamación y la fibrosis del tejido pulmonar se evaluaron mediante tinción

patológica y ensayo inmunoenzimático ligado a anticuerpos (ELISA). Los niveles

de expresión proteica de PF y de la vía TGF-β1/Smad3 se analizaron mediante

Western blot. La orientina reduce significativamente los niveles de las proteínas

TGF-β1, p-Smad3, actina alfa del músculo liso (α-SMA), colágeno I y metalopep-

tidasa de la matriz (MMP)-2. Además, el tratamiento con orientina disminuyó

significativamente las células Edu positivas, la proliferación y la migración de las

células HFL1, así como la formación de fibras de colágeno teñidas de rojo. El tra-

tamiento con orientina redujo el deterioro de la cavidad alveolar, el infiltrado de

células inflamatorias y la hiperplasia de fibras colágenas en el tejido pulmonar

de los ratones, y disminuyó significativamente el contenido de Hydroxyproline

(HYP) y de los factores inflamatorios (TNF-α e IL-6) en el lavado alveolar. Sin

embargo, SRI-011381 atenuó el efecto de la intervención con orientina, con un

aumento significativo de los niveles de inflamación y fibrosis; el SB431542 lo

acentuó. La orientina inhibe la vía de señalización TGF-β1/Smad3, la transición

de fibroblastos a miofibroblastos (FMT) y la producción de matriz extracelular

(ECM), y alivia el daño inflamatorio y fibroso.

Received: 05-11-2025 Accepted: 21-12-2025

INTRODUCTION

The main pathological features of idio-

pathic pulmonary fibrosis (IPF) are destruc-

tion of the lung parenchyma and accumu-

lation of extracellular matrix (ECM) in the

pulmonary interstitial and alveolar spaces,

which eventually leads to destruction of al-

veolar structure and severe impairment of

lung function, ultimately resulting in death1.

The median survival time of IPF patients af-

ter diagnosis is short, and the prognosis is

very poor 2, 3. At present, anti-pulmonary fi-

brosis (PF) drugs and lung transplantation

are the main treatment options for PF. Lung-

derived problems limit lung transplantation.

Orientin reduces pulmonary fibrosis 243

Vol. 67(2): 241 - 259, 2026

pirfenidone and nintedanib are the main

anti-fibrosis drugs. Studies have shown that

pirfenidone and nintedanib slow the decline

in lung function in IPF, but overall efficacy is

limited, with issues such as significant side

effects, high costs, and no improvement in

survival rate 3. Thus, it is vital to find safe

and effective new drugs for IPF therapy.

The pathogenesis of IPF is highly com-

plex and involves multiple mechanisms,

including the inflammatory response, oxi-

dative stress, epithelial-mesenchymal tran-

sition (EMT), and inhibition of autophagy.

These mechanisms are intertwined and

interact to drive IPF progression. Among

them, the transforming growth factor-β1

(TGF-β1)/suppressor of mother against de-

capentaplegic 3 (Smad3) signaling pathway

is important in regulating the PF process 4-7.

TGF-β1 is a core regulator of IPF and can

promote fibrosis 8. When TGF-β1 binds to its

receptor, it can phosphorylate Smad2/3 in

the cytoplasm, which in turn binds Smad2/3

to Smad4 to form a trimer, enters the nucle-

us, and binds to specific DNA sites, there-

by regulating the expression of a series of

downstream fibrosis-related genes, up-regu-

lating alpha-smooth muscle actin (α-SMA)

level, promoting the abnormal deposition

of fibrosis-related proteins, and increasing

and promoting fibroblast proliferation. It

drives the fibroblast-to-myofibroblast transi-

tion (FMT), a classical pathway of TGF-β1-

mediated fibrosis 9. Therefore, TGF-β1/Smad

signaling can regulate the transcription

and protein expressions of target genes, in-

duce PF 10. Inhibition of the TGF-β1/Smad3

pathway reduces PF. Sinomenine alleviates

inflammatory response and reverses EMT

through suppressing the TGF-β1/Smad3

pathway, thereby reducing PF 5; inhibition of

AHNAK2 via suppressing the TGF-β1/Smad3

pathway regulates EMT and alleviates PF11;

PM2.5 enhances endoplasmic reticulum

stress-induced autophagy, thereby activat-

ing the TGF-β1/Smad3 pathway, promoting

ECM overproduction, ultimately aggravating

PF 12. In summary, the TGF-β1/Smad3 is a

classic pathway that mediates organ fibrosis

and dominates the progression of fibrotic

diseases 13.

As research into treating PF with herbal

medicine deepens, monomers of herbal med-

icine have attracted considerable attention

for their single-component nature, stable

structures, and notable effects. Orientin is

a natural flavonoid widely found in many

medicinal plants, such as Trollius chinensis

Bunge, Odontosoria chinensis J. Sm., and

bamboo leaves. Modern pharmacological

studies have shown that orientin has many

biological properties, including anti-inflam-

matory, antioxidant, anti-aging, antibacte-

rial, hepatoprotective, neuroprotective, and

cardioprotective effects 14, 15. Studies have

shown that Orientin is a possible anti-inflam-

matory drug. Orientin can improve mito-

chondrial homeostasis, inhibit chondrocyte

senescence and inflammation, and reduce

osteoarthritis 16. The lung is the primary tis-

sue for orientin distribution, and orientin

has been shown to alleviate acute lung injury

in mice by exerting anti-inflammatory and

antioxidant effects 17. Therefore, we believe

that orientin may be an ideal anti-PF candi-

date drug. However, the therapeutic effect of

orientin on PF and its molecular mechanism

have not been systematically elucidated.

To evaluate the therapeutic potential

of orientin in PF, the effects of orientin on

FMT, fibrosis, inflammatory response, and

the TGF-β1/Smad3 signaling pathway were

assessed, with pirfenidone as a positive con-

trol. This study aims to demonstrate that

orientin can reduce PF through the TGF-β1/

Smad3 signaling pathway, thereby providing

an important experimental and theoretical

basis for the development of safe, effective,

and economical anti-PF natural drugs.

METHODS

Cell culture and modeling

Human embryonic lung fibroblasts

HFL1 were purchased from Procell Life Tech-

nology Co., Ltd. (CL-0106, Wuhan, China).

244 Liu and Lu

Investigación Clínica 67(2): 2026

HFL1 cells were cultured with HFL1 cell-

specific culture medium (CM-0106, Procell,

Ham’s F-12K medium containing 1% peni-

cillin-streptomycin and 10% fetal bovine se-

rum) in a cell culture box (MCO-18 AICUUV,

PHCbi, Japan) at 37℃ and 5% CO2. The sub-

culture medium was changed every 3 days.

HFL1 cells (1×105 cells/well) in the

logarithmic growth phase and in good con-

dition were inoculated into 6-well plates

and routinely cultured. The next day, after

adherence, orientin (S9009, Selleck, Shang-

hai, China) at 0, 10, 20, 40, 80, and 160 μM

was added, and cells were incubated for 24 h.

Then, 10 μL of Cell Counting Kit-8 (CCK-8)

solution (C0037, Beyotime, Shanghai, Chi-

na) was added, and cells were incubated for

2 h. The optical density (OD) at 450 nm was

measured using a microplate reader (MUL-

TISKAN, Perkin Elmer, United States), and

the cell survival rate was calculated to deter-

mine the safe concentration of orientin.

HFL1 cells were divided into the con-

trol group, the model (TGF-β1) group, the

positive control (TGF-β1+pirfenidone)

group, the orientin low, medium, and high

concentration (TGF-β1+orientin-L, TGF-

β1+orientin-M, TGF-β1+orientin-H) group,

the TGF-β1/Smad3 pathway agonist (TGF-

β1+orientin+SRI-011381) group, and the

inhibitor (TGF-β1+orientin+SB431542)

group. In the TGF-β1 group, 10 ng/mL

TGF-β1 (100-21, PeproTech, USA) was added

to stimulate HFL1 cells for 24 h 4, 5. After

that, 10 μM pirfenidone (Y159865, Beyo-

time) was added to the TGF-β1+pirfenidone

group to culture HFL1 cells for 24 h; HFL1

cells were cultured with 10, 20, and 40 μM

orientin for 24 h in orientin low, medium,

and high concentration groups, respectively.

TGF-β1+orientin+SRI-011381 and TGF-

β1+orientin+SB431542 groups were added

with 10 μM SRI-011381 (Y112753, Beyo-

time) and SB431542 (SF7890, Beyotime) at

the same time as orientin. Then CCK-8 was

used to measure the OD at 450 nm and cal-

culate cell viability.

Edu staining

After intervention, HFL1 cells were in-

cubated with an equal volume of 20 μM Edu

working solution (C0071S, Beyotime) for 3

h. The cells were fixed with paraformalde-

hyde, permeabilized with 1 mL of permeabi-

lization solution (P0097, Beyotime) for 10

min, and incubated with 0.5 mL of Click re-

action solution in the dark for 30 min. The

cells were stained with DAPI reagent and

mounted with anti-fluorescence quenching

mounting agent (0100-01, South biotech,

USA). Three fields of view were selected for

each well under the fluorescence micro-

scope (ECLIPSE TI2-A, Nikon, Japan), and

Edu-positive cells were counted using Image

J software.

Scratch healing experiment

HFL1 cells were inoculated into 6-well

plates and cultured overnight in serum-free

medium after reaching confluence. A 200

μL pipette tip was used to create vertical

scratches in each well, and the culture me-

dium was replaced. The cells were then sub-

jected to the experimental interventions and

cultured for 24 h. Images were taken under a

microscope (NIKON ECLIPSE E100, Nikon)

at the beginning and end of the culture, and

the distance between scratches was mea-

sured using ImageJ software to calculate the

scratch-healing rate.

Sirius red staining

After the HFL1 cells were cultured, 100

μL of ice-cold methanol was added to fix the

cells for 5 h, and then the ice-cold methanol

in the well was discarded. Hematoxylin was

added to each well for staining for 20 min,

and then 200 μL of Sirius red staining solu-

tion (GC307014, Servicebio, Wuhan, China)

was added. The cells were incubated in the

dark for 4 h. Images were observed and col-

lected under a microscope. The collagen

fibers appeared red, and the muscle fibers

appeared yellow. The area of collagen fibers

was analyzed using Image J software.

Orientin reduces pulmonary fibrosis 245

Vol. 67(2): 241 - 259, 2026

Immunofluorescence

HFL1 cells were fixed with 4% parafor-

maldehyde for 15 min, incubated with 100

μL of membrane-breaking working solution

for 10 min, blocked with 3% BSA (bovine

serum albumin) for 30 min, and incubated

with the α-SMA antibody (67735-1-Ig, Pro-

teintech, Wuhan, China) overnight at 4°C.

Alexa 488-labeled fluorescent secondary an-

tibody diluent (GB25303, Servicebio) was

added, and the mixture was incubated for 1

h in the dark. DAPI (4′,6-diamidino-2-phe-

nylindole) staining solution was then added,

and the mixture was incubated for 10 min

in the dark. Images were acquired using a

fluorescence microscope, and average fluo-

rescence intensity was analyzed with ImageJ

software.

Animal grouping, modeling,

and administration

A total of 48 SPF C57BL/6J mice, aged

8 weeks and weighing (20±2) g, were pur-

chased from Sibefu Biotechnology Co., Ltd.

(Beijing, China). The mice were housed in

clean feeding cages (ambient temperature

20-24℃, relative humidity 50%-70%, light

and dark alternating 12 h each), had free

access to food and water, and strictly ad-

hered to the ‘3R’ principle for experimen-

tal animals. All animal experimental proce-

dures were approved by the Fourth Affiliated

Hospital of China Medical University Ethics

Committee.

The mice were adaptively fed for 1 week.

They were randomly assigned to the Sham

group, the model (bleomycin, BLM) group,

the positive control (bleomycin+pirfenidone)

group, the orientin low-, medium-,

and high-dose (bleomycin+orientin-L,

bleomycin+orientin-M, bleomycin+orientin-

H) groups, the TGF-β1/Smad3 pathway agonist

(bleomycin+orientin+SRI-011381) group,

and the inhibitor (bleomycin+orientin+

SB431542) group using an interval random

grouping method based on body weight,

with 6 mice in each group. Except for the

Sham group, mice in the other groups were

anesthetized with an intraperitoneal injec-

tion of 2% pentobarbital sodium (30 mg/

kg) and then secured to the operating table.

The mouse pulmonary fibrosis model 4 was

prepared by noninvasive tracheal instillation

of bleomycin (BLM) (3.75 mg/kg, dissolved

in normal saline), and the Sham group re-

ceived the same volume of normal saline by

the same route. After 24 h of modeling, mice

in the bleomycin+pirfenidone group were

administered 100 mg/kg pirfenidone by ga-

vage. Mice in the low-, medium-, and high-

dose groups received 10, 20, and 40 mg/kg

of orientin by gavage, respectively. Mice in

the SRI-011381 and SB431542 groups were

injected intraperitoneally with 30 mg/kg

SRI-011381 and 10 mg/kg SB431542, re-

spectively, once daily for 28 days. Two hours

after the last administration, the mice were

anesthetized and sacrificed, and their lung

tissues were dissected.

Western blot

After HFL1 cells were cultured, RIPA

lysis buffer (P0013B, Beyotime) was added

to lyse the cells, and the supernatant was

collected after centrifugation (4℃, 10 000

r/min, 10 min). Mouse lung tissue was cut

into small pieces and placed in a homoge-

nizer tube. Homogenization beads and RIPA

lysate were added, and the mixture was lysed

on ice for 30 min. The supernatant was col-

lected after centrifugation (4℃, 12 000 r/

min, 10 min). The BCA protein concentra-

tion assay kit (P0009, Beyotime) was used to

determine the total protein concentration in

the supernatant. Loading buffer was added,

and the protein was denatured in a metal

bath at 96℃ for 10 min. SDS-PAGE (Sodium

Dodecyl Sulfate Polyacrylamide Gel Electro-

phoresis) was used to separate the protein

components (voltage 70-120 V, time 1.5

h). The protein was transferred to a PVDF

(polyvinylidene fluoride) membrane by wet

transfer (constant current, 300 mA; 2 h),

blocked with 5% skimmed milk powder for

2 h, and washed with PBST (phosphate-buff-

ered saline) three times. The primary anti-

246 Liu and Lu

Investigación Clínica 67(2): 2026

bodies were added and incubated overnight

at 4℃. The secondary antibody (ab6734, ab-

cam) was added and incubated for 2 h. The

chemiluminescence imaging system (Chemi

Doc MP, BioRad, USA) was used to expose

and capture images, and the gray value of

protein bands was analyzed using Image J

software.

The primary antibodies: TGF-β1

(81746-2-RR, Proteintech), Smad3 (66516-

1-Ig, Proteintech), p-Smad3 (ab63403, Ab-

cam), α-SMA (67735-1-Ig, Proteintech),

fibronectin (ab2413, Abcam), Collagen I

(ab316222, Abcam), Vimentin (ab20346,

Abcam), matrix metallopeptidase (MMP)-2

(ab181286, Abcam), MMP-9 (ab58803, Ab-

cam), and GAPDH (ab181603, Abcam) were

diluted to 1:1,000.

Pathological examination of lung tissue

The left lung tissues of mice were

fixed in 4% paraformaldehyde, dehydrated,

cleared, paraffin-embedded, and sectioned

(4 μm thickness). Hematoxylin and eosin

(HE) and Masson staining were performed

routinely. Five fields of view were selected for

each section. The pathological morphology

of lung tissue was observed under an optical

microscope, and images were collected. The

collagen deposition area in Masson-stained

sections was analyzed using ImageJ software.

Lung tissue inflammation scores were

assessed according to the Szapiel scoring

criteria: 0 score—No alveolar inflammation;

1 score—Monocyte infiltration, widened

alveolar septum, limited to local and near-

pleural areas, with an area less than 20% of

the whole lung, and normal alveolar struc-

ture; 2 score—The affected area accounted

for 20%-50% of the lung, with the area near

the pleura more severe; 3 score—Alveolitis

area >50%, with occasional consolidation

caused by monocytes and hemorrhage in the

alveolar cavity 18.

Pulmonary fibrosis was scored accord-

ing to the Ashcroft scoring standard: 0

score—normal lung tissue; 1 score—slight

thickening of the alveolar or bronchial

walls; 3 score—moderate thickening of the

alveolar or bronchial walls, with no obvious

damage to the alveolar structure; 5 score—

fibrous bands or small fibrous foci were

formed, and the alveolar structure was mark-

edly destroyed; 7 score—alveolar structure

was severely deformed, and extensive fibrous

foci were formed, showing ‘honeycomb

lung’; 8 score—full-field fibrosis of lung tis-

sue; the severity of the lesions in the 2, 4,

and 6 scores was between the corresponding

scores 19.

Bronchoalveolar lavage fluid (BALF)

analysis

After the mice were sacrificed, the

chest cavity was opened and the heart re-

moved. The lungs were washed twice with

pre-cooled normal saline. The left pulmo-

nary hilum was carefully ligated with a sur-

gical suture, and the trachea was separated

at the neck. A V-shaped incision was made

at the tracheal bifurcation, and a venous in-

dwelling needle cannula was inserted into

the lower bronchus. Then, 500 μL of normal

saline was slowly injected. Lung swelling

was visible to the naked eye, and the lavage

fluid was slowly withdrawn. After 3 repeated

injections of bronchoalveolar lavage fluid,

BALF was collected in a sterile tube, and the

supernatant was collected after centrifuga-

tion. Tumor necrosis factor-alpha (TNF-α),

interleukin (IL)-2, and IL-6 (ab208348,

ab100706, ab222503, Abcam) levels in

BALF were measured using enzyme-linked

immunosorbent assay (ELISA) kits accord-

ing to the manufacturer’s instructions. At

the same time, the activity of myeloperoxi-

dase (MPO, ab275109, Abcam) in lung tis-

sues was assessed. The total protein content

in BALF was measured using a BCA protein

concentration assay kit.

Determination of hydroxyproline (HYP)

level

According to the HYP kit (A030-2-1,

Jiancheng Institute of Bioengineering, Nan-

jing, China), 30-100 mg of wet-weight lung

Orientin reduces pulmonary fibrosis 247

Vol. 67(2): 241 - 259, 2026

tissue was accurately weighed and placed in

a test tube. 1 mL of hydrolysate was added,

and the mixture was mixed. The mixture was

incubated in a 95°C water bath for 20 min,

then diluted with double-distilled water to

10 mL. 4 mL of the diluted hydrolysate was

mixed with an appropriate amount of acti-

vated carbon, then centrifuged for 10 min.

The OD value of each group was measured

using 1 mL of supernatant at 550 nm. HYP

content in mice was calculated.

Immunohistochemistry

Lung sections were dewaxed, rehydrat-

ed, and antigen-repaired using citrate buffer.

Endogenous peroxidase was quenched with

3% H2O2 for 10 min and blocked with 5% BSA

for 20 min. Sections were incubated with the

α-SMA antibody (67735-1-Ig, Proteintech,

Wuhan, China) overnight at 4°C, then with

the secondary antibody solution for 30 min.

After washing, DAB (3,3′-diaminobenzidine)

staining was performed, and the nuclei were

counterstained with hematoxylin. α-SMA

staining was brown, and the sections were

evaluated under an optical microscope.

Statistical analysis

SPSS 27.0 was used for statistical analy-

sis. All data were tested for normality and ho-

mogeneity of variance. One-way ANOVA with

Tukey’s post hoc test was used to compare

groups. Data for each group were expressed

as mean ± standard deviation. p<0.05 was

considered statistically significant.

RESULTS

Orientin inhibited TGF-β1-activated HFL1

cell proliferation and migration

TGF-β1 was used to stimulate HFL1

cells to assess the therapeutic effect of ori-

entin on PF, and pirfenidone served as a

positive control. The structural formula of

orientin is shown in Fig. 1A. First, at con-

centrations of ≥ 80 μM, the survival rates

of HFL1 cells were markedly reduced (Fig.

1B), indicating cellular damage at this con-

centration. Therefore, 10, 20, and 40 μM

orientin were selected for subsequent ex-

periments. HFL1 cells were then stimulated

with TGF-β1 and treated with pirfenidone

and orientin. The proliferation rate of HFL1

cells increased markedly after TGF-β1 stimu-

lation but decreased significantly after pir-

fenidone and orientin treatment (Fig. 1C).

Edu staining showed that Edu-positive cells

rose notably after TGF-β1 stimulation and

decreased significantly after pirfenidone and

orientin treatment (Fig. 1D), indicating that

TGF-β1 promoted HFL1 cell proliferation,

whereas orientin inhibited this proliferation.

TGF-β1 induced the transformation of HFL1

cells into myofibroblasts, and the migra-

tion ability of myofibroblasts was enhanced

compared with fibroblasts. The migration

rate was significantly elevated after TGF-β1

stimulation at 24 h. However, after treat-

ment with pirfenidone and orientin, the mi-

gration rate was significantly reduced (Fig.

1E), indicating that orientin suppressed the

migratory ability of HFL1 cells. These exper-

iments showed that pirfenidone and orientin

significantly suppressed HFL1 cell growth

and migration, and the inhibitory effect of

high-dose orientin was comparable to that of

pirfenidone.

Orientin inhibited the TGF-β1-activated

TGF-β1/Smad3 pathway and inhibited cell

proliferation and migration in HFL1 cells

The TGF-β1/Smad3 pathway is a well-

characterized pathway implicated in PF pro-

gression. This study also found that TGF-β1

and p-Smad3 protein expression in HFL1

cells increased significantly after TGF-β1

induction but decreased significantly after

pirfenidone and orientin intervention (Fig.

2A-C), indicating that TGF-β1 induced acti-

vation of this pathway, whereas orientin in-

hibited its activation. Since the inhibitory

effect of 40 μM orientin was the most signifi-

cant in previous studies, this concentration

was selected for subsequent research. HFL1

cells were treated with the TGF-β1/Smad3

pathway agonist SRI-011381 and the inhibi-

248 Liu and Lu

Investigación Clínica 67(2): 2026

tor SB431542 alongside orientin treatment.

Compared with orientin treatment, TGF-β1

and p-Smad3 protein expression increased

significantly after SRI-011381 treatment

and decreased significantly after SB431542

treatment (Fig. 2D-F). After SRI-011381 in-

tervention, the proliferation rate of HFL1

cells (Fig. 2G) and Edu-positive cell number

(Fig. 2H) increased significantly, and the

migration rate also increased significantly

(Fig. 2I). SB431542 significantly reduced

the proliferation rate, Edu-positive cell num-

Fig. 1. Orientin inhibited TGF-β1-induced HFL1 cell growth and migration. A: Orientin molecular formula. B:

HFL1 (cells derived from human fetal lung tissue) were treated with orientin for 24 h, and the survival

rate was measured by CCK-8 assay (Cell Counting Kit-8). Orientin at concentrations of 40 μM or lower

had no effect on cell viability (*p<0.05, **p<0.01 vs 0 group). C: After HFL1 cells were stimulated

with TGF-β1, they were treated with pirfenidone and orientin. The proliferative ability of HFL1 cells

was assessed by the CCK-8 assay. Orientin markedly inhibited cell proliferation. D: HFL1 cell prolife-

ration was assessed by EdU staining (5-ethynyl-2′-12 deoxyuridine). Orientin decreased the number

of EdU-positive cells (×40, 50 μm). E: The migration of HFL1 cells was assessed by the wound-healing

assay. Orientin significantly inhibited cell migration (×10, 200 μm). 15 n=3, *p<0.05, ** p<0.01,

*** p<0.001 vs 0/Control group; ## p<0.01, ### p<0.001 vs TGF-β1 group.

Orientin reduces pulmonary fibrosis 249

Vol. 67(2): 241 - 259, 2026

ber, and migration rate of HFL1 cells (Fig.

2G-I), indicating that inhibition of TGF-β1/

Smad3 signaling could inhibit HFL1 cell pro-

liferation and migration. In conclusion, ori-

entin suppressed the TGF-β1/Smad3 path-

way and inhibited HFL1 cell proliferation

and migration.

Fig. 2. Orientin inhibited TGF-β1-activated TGF-β1/Smad3 pathway and inhibited cell proliferation and migra-

tion in HFL1 cells. A-C: Western blot was used to detect the expression of the TGF-β1/Smad3 pathway

protein in HFL1 cells (cells derived from human fetal lung tissue). Expression of TGFβ1and p-Smad3 pro-

tein increased significantly after TGF-β1 induction, and decreased significantly after orientin interven-

tion. D-F: HFL1 cells were treated with the TGFβ1/Smad3 pathway agonist SRI-011381 (N′-cyclohexyl-

N-(phenylmethyl)-N-(4piperidinylmethyl)-urea) and inhibitor SB431542 (TGF-β RI Kinase Inhibitor VI)

alongside orientin treatment, and TGF-β1/Smad3 pathway protein levels were detected through Wes-

tern blot. TGF-β1 and p-Smad3 protein expressions increased significantly after SRI-011381 treatment,

and decreased significantly after SB431542 treatment. G: HFL1 cell proliferation was determined by

CCK-8 (Cell Counting Kit-8). SB431542 markedly inhibited cell proliferation. H: HFL1 cell proliferation

was assessed by EdU staining (5-ethynyl-2′-deoxyuridine). SB431542 significantly reduced the number

of EdU-positive cells (×40, 50 μm). I: Migration was evaluated by the scratch-healing assay. SB431542

significantly inhibited cell migration (×10, 200 μm). n=3, *** p<0.001 vs Control group; #p<0.05,

##p<0.01, ###p<0.001 vs TGF-β1 group; &&p<0.01, &&& p<0.001 vs TGF-β1+Orientin group.

250 Liu and Lu

Investigación Clínica 67(2): 2026

Orientin inhibited TGF-β1-induced

fibroblast to myofibroblast transition

(FMT) and improved extracellular matrix

(ECM) through the TGF-β1/Smad3

signaling pathway

Fibroblasts are primarily responsible

for producing and maintaining the ECM, and

their mechanical properties can be altered.

They can also transform into myofibroblasts.

Myofibroblasts are cells that drive fibrotic

tissue development through a sharp increase

in protein deposition. The transition from fi-

broblasts to myofibroblasts is a well-known

cellular marker of histopathological status 20.

Sirius red staining showed that collagen fiber

staining increased significantly after TGF-β1

stimulation and decreased significantly after

orientin treatment. However, compared with

orientin treatment, red-stained collagen fi-

bers increased significantly after SRI-011381

treatment and decreased significantly after

SB431542 treatment (Fig. 3A), indicating

that orientin inhibited the production and

accumulation of collagen fibers induced by

TGF-β1 through the TGF-β1/Smad3 path-

way. α-SMA is a marker of myofibroblasts.

The fluorescence intensity of α-SMA in-

creased significantly after TGF-β1 interven-

tion, decreased significantly after orientin

treatment, increased significantly after SRI-

011381 treatment, and decreased signifi-

cantly after SB431542 treatment (Fig.3B).

Finally, ECM-related protein expression was

assessed by Western blot. α-SMA, fibronec-

tin, Collagen I, Vimentin, MMP-2, and MMP-

9 protein levels were markedly elevated after

TGF-β1 stimulation but declined markedly

after orientin treatment. Compared with

orientin intervention, protein levels were

significantly increased after SRI-011381

treatment and significantly decreased after

SB431542 treatment (Fig. 3C-I), indicat-

ing that orientin improved ECM through

the TGF-β1/Smad3 pathway. Overall, these

experiments showed that orientin inhibited

TGF-β1-induced FMT, reduced ECM deposi-

tion, and promoted ECM remodeling by sup-

pressing the TGF-β1/Smad3 pathway.

Orientin inhibited the BLM-activated

TGF-β1/Smad3 pathway in mouse lung

tissue

To further verify the protective effects

of orientin in vivo, this study established a

BLM-induced PF mouse model to verify the

in vivo mechanism and treated the mice

with pirfenidone and orientin. TGF-β1 and

p-Smad3 protein expression increased mark-

edly after BLM induction but decreased

significantly after pirfenidone and orientin

treatment, with the high-dose orientin in-

tervention effect comparable to that of pir-

fenidone (Fig. 4A-C). However, after injec-

tion of SRI-011381, TGF-β1 and p-Smad3

levels were notably raised. The protein levels

were significantly reduced after injection

of SB431542 (Fig. 4D-F). This indicated

that orientin suppressed the BLM-induced

TGF-β1/Smad3 pathway.

Orientin alleviated BLM-induced

inflammatory injury in mouse lung tissue

by the TGF-β1/Smad3 pathway

HE staining showed that the lung tis-

sue structure in the bleomycin group was

destroyed, with alveolar cavities collapsed,

alveolar septa widened, and inflammatory

infiltration within the alveolar cavities. The

inflammatory score of the lung tissue in-

creased significantly. After orientin treat-

ment, inflammatory infiltration and the

score were significantly reduced. After SRI-

011381 treatment, the alveolar space in

lung tissue decreased, inflammatory infil-

tration increased, and the score increased.

Lung tissue inflammation was inhibited after

SB431542 treatment (Fig. 5A). TNF-α, IL-2,

and IL-6 levels in BALF, and MPO (myelo-

peroxidase) activity in lung tissue, increased

significantly after BLM stimulation and de-

creased significantly after orientin treat-

ment. Compared with orientin treatment,

inflammatory factors and MPO levels were

significantly increased after SRI-011381

treatment and significantly decreased after

SB431542 treatment (Fig. 5B-E). The trend

of total protein content in BALF was consis-

Orientin reduces pulmonary fibrosis 251

Vol. 67(2): 241 - 259, 2026

tent with that of inflammatory factors, in-

creasing significantly after treatment with

BLM and SRI-011381, and decreasing sig-

nificantly after treatment with orientin and

SB431542 (Fig.5F). In summary, orientin

can alleviate the degree of inflammation in

PF mice by suppressing the TGF-β1/Smad3

pathway.

Fig. 3. Orientin inhibited TGF-β1-induced FMT and improved ECM by TGFβ1/Smad3 pathway. A: Sirius red

staining was used to detect the type of cell collagen fibers. Red-stained collagen fibers were signi-

ficantly reduced after Orientin treatment (×20, 100 μm). B: α-SMA levels were assessed by immu-

nofluorescence. Orientin significantly reduced α-SMA fluorescence intensity (×40, 50 μm). C-I: ECM

(extracellular matrix)-related protein expression was determined by Western blot. α-SMA, fibronectin,

Collagen I, Vimentin, MMP-2 (matrix metalloproteinase-2), and MMP-9 (matrix metalloproteinase-9)

levels were markedly decreased after Orientin therapy. n=3, *** p<0.001 vs Control group; ### p

<0.001 vs TGF-β1 group; && p<0.01, &&& p<0.001 vs TGF-β1+Orientin group. TGF-β1.

252 Liu and Lu

Investigación Clínica 67(2): 2026

Orientin alleviated BLM-induced PF

injury in mice through the TGF-β1/Smad3

pathway

HYP content in lung tissue increased

significantly after BLM stimulation and de-

creased significantly after orientin treat-

ment. Compared with orientin treatment,

HYP content increased significantly after

SRI-011381 treatment and decreased sig-

nificantly after SB431542 treatment (Fig.

6A). Masson staining showed that the struc-

ture of lung tissue in the bleomycin group

was destroyed, collagen fibers were se-

verely proliferated, and the collagen deposi-

tion area and pulmonary fibrosis score were

significantly increased. After orientin treat-

ment, the degree of alveolar damage and

collagen fiber hyperplasia were significantly

reduced, and the collagen deposition area

and pulmonary fibrosis score were signifi-

cantly reduced. After the SRI-011381 inter-

vention, collagen fiber proliferation was no-

tably elevated, and the collagen deposition

area and PF score were markedly increased.

After SB431542 treatment, collagen fiber

proliferation was reduced, and the collagen

deposition area and PF score were notably

reduced (Fig.6B-D). α-SMA levels were con-

sistent with those in cell experiments, being

markedly raised after BLM and SRI-011381

treatment and significantly decreased after

orientin and SB431542 treatment (Fig. 6E).

Fibronectin, Collagen I, α-SMA, Vimentin,

MMP-2, and MMP-9 expressions increased

Fig. 4. Orientin inhibited BLM-induced activation of the TGF-β1/Smad3 pathway 65 in mouse lung tissue. A-C:

Protein expression of the TGF-β1/Smad3 pathway was evaluated by Western blot. TGF-β1 and p-Smad3

protein (phosphorylated SMAD family member 3) levels were markedly increased after BLM induction,

and significantly lowered after orientin intervention. D-F: The mice were injected with the TGF-β1/

Smad3 pathway agonist SRI-011381(N′-cyclohexyl-N-(phenylmethyl)-N-(4-piperidinylmethyl)-urea) and

the inhibitor SB431542 (TGF-β RI Kinase Inhibitor VI) at the same time as orientin, and TGF-β1/

Smad3 pathway protein levels were determined by Western blot. p-Smad3 (phosphorylated SMAD family

member 3) and TGF-β1 levels were significantly increased when SRI-011381 intervention, and signifi-

cantly decreased after SB431542 treatment. n=6, ***p<0.001 vs sham group; # p<0.05, ##p<0.01,

###p<0.001 vs treatment. n=6, ***p<0.001 vs sham group; #p<0.05, ##p<0.01, ### p<0.001 vs.

Orientin reduces pulmonary fibrosis 253

Vol. 67(2): 241 - 259, 2026

markedly after BLM stimulation but de-

creased significantly after orientin treat-

ment. Compared with orientin intervention,

protein levels were significantly increased af-

ter SRI-011381 treatment and significantly

decreased after SB431542 treatment (Fig.

6F-L). These experiments showed that orien-

tin could alleviate fibrosis damage in PF mice

by suppressing the TGF-β1/Smad3 pathway.

DISCUSSION

This study was the first to systematical-

ly evaluate the therapeutic effect of orientin

on PF and its underlying molecular mecha-

nism. Orientin notably reduced pathological

changes in lung tissue, inhibited FMT, im-

proved ECM, and lessened the severity of PF.

The mechanism was closely associated with

reducing the inflammatory response and in-

hibiting the TGF-β1/Smad3 pathway.

Myofibroblasts are absent from nor-

mal lung tissue but are important produc-

ers of collagen and other matrix proteins.

Promoting fibroblast apoptosis and inhibit-

ing fibroblast-to-myofibroblast transforma-

tion can effectively inhibit PF 21. TGF-β1, a

key cytokine that promotes myofibroblast

Fig. 5. Orientin alleviated BLM-induced inflammatory injury in mouse lung tissue via the TGF-β1/Smad3

pathway. A: Pathological damage to lung tissue in each group was observed by HE staining. After

BLM stimulation, alveolar cavity collapse, inflammatory cell infiltration, and inflammatory score in-

creased significantly. Orientin can inhibit lung inflammation (×20, 100 μm). B-E: TNF-α, IL-2 and

IL-6 levels in BALF and MPO activity in lung tissues were determined using an ELISA kit. These

were significantly reduced after Orientin treatment. F: The total protein content in BALF was de-

termined by the BCA (bicinchoninic acid) method, which was significantly decreased after Orientin

treatment. n=6, *** p<0.001 vs sham group; ###p<0.001 vs the bleomycin group; &&& p<0.001 vs

the bleomycin+orientin group. SRI-011381(N′-cyclohexyl-N (phenylmethyl)-N-(4-piperidinylmethyl)-

urea); SB431542 (TGF-β RI Kinase Inhibitor VI).

254 Liu and Lu

Investigación Clínica 67(2): 2026

Fig. 6. Orientin alleviated BLM-induced PF injury in mice through the TGF-β1/Smad3 pathway. A: HYP content

in lung tissue was measured by HYP assay, and was significantly reduced after orientin treatment. B-D:

Collagen deposition and fibrosis were analyzed by Masson-Brüch staining. After BLM stimulation, collagen

fibers in lung tissue proliferated significantly, and the collagen deposition area and PF score increased sig-

nificantly. The collagen deposition area and pulmonary fibrosis score were markedly reduced after orientin

therapy (×20, 100 μm). E: α-SMA) level was evaluated by immunohistochemistry. Orientin significantly

reduced α-SMA level (×20, 100 μm). F-L: PF-related protein levels were detected by Western blot. The levels

of α-SMA, fibronectin, Collagen I, Vimentin, MMP-2, and MMP-9 were markedly decreased after Orientin

intervention. n=6, *** p<0.001 vs the sham group; ### p<0.001 vs the bleomycin group; & p<0.05,

&&p<0.01, &&&p<0.001 vs the bleomycin+orientin group. SRI-011381(N′-cyclohexyl-N-(phenylmethyl)-

N-(4-piperidinylmethyl)-urea); SB431542 (TGF-β RI Kinase Inhibitor VI).

Orientin reduces pulmonary fibrosis 255

Vol. 67(2): 241 - 259, 2026

differentiation and collagen expression, is

vital for the progression of PF. As our un-

derstanding of PF pathogenesis advances,

TGF-β1 has been identified as the primary

pro-fibrotic growth factor involved in fi-

brosis, influencing cell growth, differentia-

tion, and programmed cell death. Excess

TGF-β1 results in collagen accumulation

and triggers various fibrotic conditions 22.

Therefore, this study used TGF-β1 to in-

duce fibroblasts and construct an in vitro

PF model. The results showed that orientin

significantly suppressed TGF-β1-activated

proliferation and migration of HEL1 cells.

In this study, the anti-fibrosis mecha-

nism of orientin was further elucidated by

systematically evaluating changes in key

indicators of the TGF-β1/Smad3 pathway.

At the ligand level, TGF-β1 is the central

factor that initiates and maintains fibrosis,

and its activity directly determines the se-

verity of fibrosis 23. At the level of intracel-

lular signal transduction, the phosphoryla-

tion level of Smad3 is a direct indicator of

TGF-β1 pathway activity 24. At the effector-

molecule level, α-SMA is a specific marker

of fibroblast activation; its expression di-

rectly reflects the activation state of fibro-

blasts. At the same time, ECM remodeling

is an important factor in promoting the oc-

currence and development of PF. Interven-

tion in ECM remodeling can significantly

improve excessive fibrosis in the lung 25, 26.

Collagen I and fibronectin are major ECM

components; their high expression can lead

to pulmonary interstitial collagen deposi-

tion 27, 28. MMPs are collagenolytic enzymes

that are vital to the pathophysiological pro-

cess of IPF. High expression of MMPs de-

grades the basement membrane and lung

tissue structure, collapses the alveolar cav-

ity, aggravates lung injury, and further pro-

motes the release of TGF-β1. TGF-β1, as an

activator of MMPs, promotes their synthesis

and further increases MMP expression, thus

forming a vicious circle 29.

This study found that orientin signifi-

cantly reduced TGF-β1 and p-Smad3 levels

in HEL1 cells after TGF-β1 stimulation, in-

dicating that orientin inhibited the TGF-β1/

Smad3 pathway and that this inhibition also

suppressed HEL1 cell proliferation and mi-

gration. In addition, TGF-β1 significantly

increased collagen fiber formation in HEL1

cells and upregulated α-SMA, fibronectin,

Collagen I, Vimentin, and MMP expression,

suggesting that TGF-β1 notably promoted

lung fibroblast activation, FMT, and exces-

sive ECM deposition. Orientin could inhibit

the TGF-β1/Smad3 pathway, thereby inhib-

iting lung fibroblast activation, improving

ECM, and alleviating the PF process. ECM

and collagen deposition are the key patho-

logical changes that cause PF, indicating

that Orientin improved these key pathologi-

cal changes.

PF involves a series of inflammatory

and fibrotic reactions that ultimately lead to

ECM deposition, fibroblast foci, and fibrotic

areas juxtaposed with normal lung parenchy-

ma. In the early stage of IPF, the balance of

M1/M2 macrophages is disrupted, leading

to the secretion of inflammatory mediators

and the recruitment of fibroblasts, which

proliferate continuously. TNF-α, IL-1β, IL-

6, and IL-2 are key biomarkers for assessing

the inflammatory state of lung tissue. They

jointly promote inflammatory injury and fi-

brosis in lung tissue by recruiting, activat-

ing, and expanding inflammatory cells, and

by regulating the immune response and tis-

sue repair processes in the development of

PF 3. Studies have shown that orientin can

reduce LPS-induced NF-κB phosphorylation

in cells, thereby lowering inflammatory fac-

tors and oxidative markers17. MPO activity

is a marker of neutrophil infiltration in lung

tissue 30. Neutrophils release numerous tox-

ic substances during inflammation, causing

damage to the lung parenchyma and tissue

structure. Neutrophils are key inflamma-

tory cells that drive PF 31, 32. The most com-

monly used and well-established drug for

inducing lung fibrosis in rats is BLM 33. In

this study, lung tissues after BLM interven-

tion were significantly damaged, with alveo-

256 Liu and Lu

Investigación Clínica 67(2): 2026

lar collapse, atrophy, widened intervals, and

inflammatory cell infiltration. In addition,

a large number of inflammatory cytokines

accumulated in BALF after BLM induc-

tion, and MPO activity in lung tissues was

markedly elevated. However, inflammatory

infiltration in the pirfenidone and orientin

groups was significantly reduced, and the

levels of TNF-α, IL-2, IL-6, and MPO activity

were also significantly reduced. SRI-011381

significantly weakened orientin’s anti-

inflammatory effects, whereas SB431542

significantly enhanced them. This suggests

that orientin may reduce the release of in-

flammatory factors and inflammatory infil-

tration via the TGF-β1/Smad3 axis, thereby

slowing the PF process.

After modeling with BLM, the initial

damage was primarily concentrated around

the bronchioles; on the 7th day, the distal

lung parenchyma was involved, and multi-

ple inflammatory lesions and edema were

observed in the alveolar septa. On the 14th

day, regional interstitial fibrosis appeared

in the lungs, manifested as extensive col-

lagen deposition and remodeling of alveo-

lar units. These changes are considered

to reflect similar changes in PF. Based

on this, the model can complete the task

of studying PF mitigation drugs. HYP is a

unique amino acid in collagen and is one

of the main components of collagen tis-

sue. Some data confirmed that HYP can be

used as a reliable method for quantitative

fibrosis4. In the BLM-induced PF rat model,

this study, using Masson staining, observed

that collagen fibers after BLM intervention

were markedly proliferated, the collagen

deposition area and PF score were mark-

edly increased, and the HYP content was

significantly increased. Collagen deposition

in the pirfenidone and orientin groups was

notably reduced, and the PF score and HYP

content were significantly reduced, indicat-

ing that orientin and pirfenidone can ef-

fectively alleviate BLM-induced pulmonary

fibrosis. Western blot experiments also con-

firmed that the expressions of fibronectin,

Collagen I, α-SMA, Vimentin, MMP-2, and

MMP-9 were significantly decreased after

orientin treatment. However, SRI-011381

significantly weakened orientin’s anti-fibro-

sis effect, whereas SB431542 significantly

enhanced it. Therefore, orientin can inhibit

lung fibroblast activation and reduce col-

lagen deposition in the lungs of PF mice

via the TGF-β1/Smad3 pathway. Combined

with the results of cell experiments, Orien-

tin can inhibit FMT through the TGF-β1/

Smad3 pathway, alleviate inflammatory re-

sponses and fibrotic damage, and thereby

improve PF, indicating the potential of Ori-

entin as a therapeutic drug for PF.

In summary, orientin can inhibit inflam-

matory factors and FMT in a PF model, re-

duce ECM production, regulate key protein

expression in the TGF-β1/Smad3 pathway,

and demonstrate a clear anti-fibrotic effect.

As a natural medicine with a wide range of

sources and low cost, orientin has promis-

ing applications in PF treatment. This study

provides laboratory evidence supporting the

use of orientin as a therapeutic agent for

PF. Follow-up studies can further explore its

clinical translational value and provide new

treatment options for PF patients. Although

this study reveals the potential role of orien-

tin in PF and its molecular mechanism, dif-

ferences between rodents and humans intro-

duce uncertainty in translating the results,

which can be further explored and verified

using PF patient samples.

Orientin reduces pulmonary fibrosis 257

Vol. 67(2): 241 - 259, 2026

List of abbreviations

TGF-β1 transforming growth factor-β1

Smad3 suppressor of mother against

decapentaplegic 3

HYP hydroxyproline

FMT fibroblast to myofibroblast transition

PF pulmonary fibrosis

IPF idiopathic pulmonary fibrosis

ECM extracellular matrix

EMT epithelial-mesenchymal transition

α-SMA alpha-smooth muscle actin

MMP matrix metallopeptidase

BALF bronchoalveolar lavage fluid

TNF-αtumour necrosis factor-alpha

IL interleukin

MPO myeloperoxidase

IMP Imperatorin

BLM bleomycin

Acknowledgment

None.

Funding

None.

ORCID ID of the authors

• Yijin Liu (YiL):

0009-0000-9374-5973

• Yao Lu (YaL):

0009-0007-2749-1419

Author's contributions

YaL: Developed and planned the study,

performed experiments, and interpreted

results. Edited and refined the manuscript

with a focus on critical intellectual contri-

butions. YaL: Participated in collecting, as-

sessing, and interpreting the date. Made sig-

nificant contributions to date interpretation

and manuscript preparation. Yi L: Provided

substantial intellectual input during the

drafting and revision of the manuscript.

Conflict of interest

The authors state that they have no

conflicts of interest.

Consent to publish

The manuscript has neither been previ-

ously published nor is under consideration

by any other journal. The authors have all ap-

proved the content of the paper.

Ethic approval

This study was approved by The Fourth

Affiliated Hospital of China Medical Universi-

ty Ethic Committee (No. 21000062024112).

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