Research ArticleExperimental Studies
Open Access

Silibinin Mitigates Vanadium-induced Lung Injury via the TLR4/MAPK/NF-κB Pathway in Mice

HOBIN IM, EUNGYUNG KIM, HONG JU KWON, HYEONJIN KIM, JIWON KO, YONGHUN SUNG, SUNG-HYUN KIM, EUN JUNG LEE, WOO-SUNG KWON, ZAE YOUNG RYOO, JUNKOO YI, SI JUN PARK and MYOUNG OK KIM
In Vivo September 2024, 38 (5) 2179-2189; DOI: https://doi.org/10.21873/invivo.13681

Abstract

Background/Aim: Silibinin, has been investigated for its potential benefits and mechanisms in addressing vanadium pentoxide (V2O5)-induced pulmonary inflammation. This study explored the anti-inflammatory activity of silibinin and elucidate the mechanisms by which it operates in a mouse model of vanadium-induced lung injury. Materials and Methods: Eight-week-old male BALB/c mice were exposed to V2O5 to induce lung injury. Mice were pretreated with silibinin at doses of 50 mg/kg and 100 mg/kg. Histological analyses were performed to assess cell viability and infiltration of inflammatory cells. The expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and activation of the MAPK and NF-Embedded ImageB signaling pathways, as well as the NLRP3 inflammasome, were evaluated using real-time PCR, western blot analysis, and immunohistochemistry. Whole blood analysis was conducted to measure white blood cell counts. Results: Silibinin treatment significantly improved cell viability, reduced inflammatory cell infiltration, and decreased the expression of pro-inflammatory cytokines in V2O5-induced lung injury. It also notably suppressed the activation of the MAPK and NF-Embedded ImageB signaling pathways, along with a marked reduction in NLRP3 inflammasome expression levels in lung tissues. Additionally, silibinin-treated groups exhibited a significant decrease in white blood cell counts, including neutrophils, lymphocytes, and eosinophils. Conclusion: These findings underscore the potent anti-inflammatory effects of silibinin in mice with V2O5-induced lung inflammation, highlighting its therapeutic potential. The study not only confirms the efficacy of silibinin in mitigating inflammatory responses but also provides a foundational understanding of its role in modulating key inflammatory pathways, paving the way for future therapeutic strategies against pulmonary inflammation induced by environmental pollutants.

Key Words:

Air pollution, a critical environmental issue, is exacerbated by rapid industrialization and urbanization, contributing significantly to respiratory ailments (1-3). Extensive research over the years has linked air pollution to a spectrum of respiratory illnesses, including lung cancer, asthma, and chronic obstructive pulmonary disease (4). Particulate matter (PM), a major air pollutant, exerts systemic effects that extend beyond the lungs (5-7). PM is categorized by size: PM10 includes particles smaller than 10 μm, while PM2.5 comprises finer particles smaller than 2.5 μm. PM2.5 can penetrate deep into the bronchial tubes, reaching the delicate bronchial tree and alveoli (8). Various sources contribute to PM levels, including traffic, industrial activities, construction, combustion processes, natural events, and secondary emissions, which are pollutants formed by the transformation of primary emissions in the atmosphere (9, 10). PM is composed of sulfates, nitrates, metals like vanadium pentoxide (V2O5), lead, iron, cadmium, and organic compounds such as polycyclic aromatic hydrocarbons. Studies suggest that exposure to PM of 2.5 μm or smaller adversely affects the respiratory, cardiovascular, and reproductive systems (11-15). Among these metals, V2O5 is a notable component of PM2.5 and is known for its potential to cause significant lung toxicity upon chronic exposure (16).

Previous studies have identified that V2O5 can induce oxidative stress by elevating reactive oxygen species (ROS) levels (17), leading to pulmonary edema and inflammation, particularly in workers exposed to vanadium (14, 18). Long-term exposure to V2O5 in mice has been associated with chronic lung inflammation, lung cancer, reproductive issues, and vascular inflammation (19-22). The lungs, being highly sensitive to particulate exposure, are particularly vulnerable to V2O5-induced inflammation, making this a critical research focus (23).

The MAPK and NF-Embedded ImageB signaling pathways play a pivotal role in the inflammatory response (24-27). Increased ROS levels can lead to oxidative stress, and the activation of NF-Embedded ImageB can amplify the secretion of various inflammatory cytokines, including TNF-α, IL-6, and IL-1β (28), which in turn can trigger a pulmonary inflammatory response (29). Moreover, the activation of the NLRP3 inflammasome by ROS can cause inflammation through the release of IL-1β following caspase-1 activation (30).

Compounds that modulate key signaling pathways, including NF-Embedded ImageB, MAPK, and NLRP3, are recognized for their potential to mitigate inflammatory and oxidative stress (30-33). Silibinin, derived from milk thistle seeds and known for its historical medicinal use (34, 35), has been shown to target NF-Embedded ImageB, offering anti-inflammatory and anti-cancer benefits across various organs (36-39). In vitro studies have demonstrated that silibinin can suppress the inflammatory response by down-regulating NF-Embedded ImageB and MAPK signaling (40, 41). Similarly, in vivo studies have indicated that silibinin can inhibit lung fibrosis and injury by modulating the MAPK pathway (42, 43). Despite these findings, direct evidence of silibinin’s therapeutic effects in a V2O5-induced lung inflammation mouse model is lacking. Therefore, in this study, we conducted a comprehensive investigation into the anti-inflammatory and antioxidant effects of silibinin on lung inflammation induced by V2O5.

Materials and Methods

Reagents. V2O5 (pure 98%, 181.88 MW, CAS #: 1314-62-1) and silibinin (pure 98%, 482.44 MW, CAS #: 22888-70-6) were purchased from Sigma Aldrich (St Louis, MO, USA). Gefitinib and imatinib (pure 100%, 589.7 MW, CAS #: 13166, 13139) were purchased from Cayman Chemical (Ann Arbor, MI, USA). 6-shogaol was purchased from Chengdu Biopurify (PS010913, Chengdu Push Biotechnology Co, Ltd., Chengdu, Sichuan, PR China). All experiments were performed in accordance with the ARRIVE guidelines.

Cell culture. The human lung embryo cell line, L132, was purchased from the Korea Cell Line Bank (Seoul, Republic of Korea). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin antibiotics (Gibco, Thermo Fisher Scientific).

Cell viability assay. The natural compounds (6-shogaol, silibinin, gefitinib, and imatinib) were tested to select the final candidate for a therapeutic compound for vanadium-induced lung injury. Cells were seeded into 96-well plates (1×103 cells/well) and allowed to attach overnight. The cells were then treated with various concentrations of the compounds or dimethyl sulfoxide for 0, 24, 48, 72, and 96 h. Cells were treated with various concentrations (1, 2, 5, 10, and 15 μM) of V2O5 and incubated at 37°C for 1 h. Subsequently, 10 μl CCK-8 reagent was added to each well and the cells were incubated for an additional 2 h. The absorbance was measured at 450 nm using a microplate reader. When the compounds were evaluated, the cells were treated in the same manner. The compounds were used at a concentration of 20 μM 1 h before V2O5 treatment.

Animals. Eight-week-old male BALB/c mice were utilized. Mice were acclimatized for one week before the experiment in a controlled environment with 50±10% humidity, a 12-hour light/dark cycle, and a temperature of 22±2°C. The control group was not exposed to V2O5 but received the same handling as the experimental groups. The mice were provided with tap water and weighed weekly. The Animal Testing Ethics Committee of Kyungpook National University approved this study for animal experiments (approval no. 2019-0056).

Establishment of lung injury mice. Mice were randomized into four groups (6 mice per group): 1) control group; 2) V2O5 group; 3) silibinin 50 group (silibinin 50 mg/kg+V2O5); 4) silibinin 100 group (silibinin 100 mg/kg+V2O5). Silibinin was dissolved in 100 μl of distilled water at a dose of 50 mg/kg (low dose) and 100 mg/kg (high dose). Mice were pretreated with silibinin 50 mg/kg; 100 mg/kg in 100 μl by oral administration, 1 h before V2O5 exposure. The mice were placed in a whole-body inhalation chamber (Gaon bio, Yongin, Republic of Korea), where they were exposed to particulate aerosols of V2O5 concentration of either 0 (control) or 4 mg/m3 (V2O5 group, silibinin 50 group, silibinin 100 group) for 6 h per day, three days per week for eight weeks (20, 44). The mice were sacrificed after the final exposure by cervical dislocation.

Whole blood analysis. Immediately after sacrifice, whole blood was collected from the mice and analyzed using an ADVIA 120 Hematology system (Korea Polytech College, Nonsan, Republic of Korea). Next, cell number analyses of white blood cells (WBC), neutrophils, lymphocytes, and eosinophils were performed.

Histological analysis. Mouse lung tissues were fixed with 4% formaldehyde, paraffin-embedded, and cut into 4-μm sections. The sections were stained with hematoxylin and eosin (H&E). Each section was observed using a light microscope (Olympus BX43, Olympus, Tokyo, Japan) to estimate inflammatory cell infiltration. Furthermore, the bronchial thickness in lung tissue and H scoring were determined by skilled researchers. The H score was considered 5 points if the intensity of inflammation was severe and 1 point if inflammation was weak; 1 (normal=no inflammation), 2 (minimal= perivascular, peribronchial or patchy interstitial inflammation involving <10% of lung volume), 3 (mild=perivascular, peribronchial or patchy interstitial inflammation involving 10-20% of lung volume), 4 (moderate=perivascular, peribronchial, patchy interstitial or diffuse inflammation involving 20-50% of lung volume), and 5 (severe=diffuse inflammation involving >50% of lung volume).

RNA extraction and real-time PCR. Total RNA was isolated from lung tissues using TRIzol reagent. Total RNA was converted to cDNA using the PrimeScript™ 1st strand cDNA synthesis kit (6110, Takara, Beijing, PR China). For PCR amplification, the following primers were used: mouse β-actin forward, 5′-GGC TCT TTT CCA GCC TTC CT-3′ and reverse, 5′-GTC TTT ACG GAT GTC AAC GTC ACA-3′; IL-1β forward, 5′-CCC CAG GGC ATG TTA AGG A-3′, and reverse, 5′-TGA CCC TGA GCG ACC TGT CT-3′; IL-6 forward, 5′-GTT GTG CAA TGG CAA TTC TGA-3′, and reverse, 5′-TTG GTA GCA TCC ATC ATT TCT TTG-3′; TNF-α forward, 5′-AGG ACC CAG TGT GGG AAG CT-3′, and reverse, 5′-AAA GAG Prime Script GCA ACA AGG TAG AGA-3′. The PCR reaction mixture contained 8 μl cDNA, 10 μl Power SYBR Green PCR Master Mix (4367659, Applied Biosystems, Thermo Fisher Scientific, Altrincham, UK), 1 μl 0.2 pmol forward primer, and 1 μl 0.2 pmol reverse primer. The Applied Biosystems real-time PCR program consisted of a holding stage at 95°C for 10 min, followed by 40 cycles of cycling at 95°C for 15 s, 60°C for 1 min, followed by a melt curve. The relative expression of IL-1β, IL-6, and TNF-α mRNA was normalized to that of β-actin mRNA.

Western blot analysis. Lung tissues were homogenized in tissue lysis buffer (Intron Biotechnology, Gyeonggi-do, Republic of Korea). Protein concentration was determined using the BCA Protein Assay reagent (Thermo Scientific) according to the manufacturer’s instructions. Equal amounts of protein lysate were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight with primary antibodies at 4°C. The following primary antibodies and dilutions were used: TNF-α, IL-1β, phospho-p38, total p38, phospho-JNK, total JNK, phospho-ERK1/2, total ERK1/2, phosphor-p65, total p65, NLRP3 (1:1,000 dilution; Cell signaling Technology, Danvers, MA, USA), SOD1, and SOD2 (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. Immunoblots were visualized using ECL detection kit (GE Healthcare, Seoul, Republic of Korea) by Imagequant™ LAS 500 (GE Healthcare).

Immunohistochemical analysis (IHC). NLRP3 proteins in lung tissues were examined with IHC staining. The samples were fixed with paraformaldehyde at 4°T for 4 h, washed with phosphate-buffered saline containing 20% sucrose for 4 h, embedded and cut into 4-μm-thick sections on acid pretreated slides. After dewaxing, blocking endogenous peroxidase, and repairing the antigen, the lung tissue sections were incubated with anti-rabbit NLRP3 antibodies (1:200 dilution) at 4°C overnight, followed by incubation with HRP-labeled Goat Anti-Mouse IgG (H+L) as a secondary antibody (1:100 dilution) at 37°C for 30 min. The results were observed under a light microscope.

Statistical analysis. All results are expressed as the means±standard deviation (SD) from at least three independent experiments. All analyses were performed using SPSS software (IBM SPSS Statistics 26.0, IBM Corp., Armonk, NY, USA). Normality tests were performed with Kolmogorov–Smirnov and Shapiro–Wilk test (p>0.05). Statistical significance between experimental groups was determined using one-way ANOVA for pair-wise comparisons with Dunnett’s test. p<0.05 was considered to indicate a statistically significant difference.

Results

Enhancement of human lung epithelial cell viability by silibinin in vitro. Leveraging natural compounds for the treatment of inflammatory diseases offers several benefits, including reduced treatment costs and side effects for patients. In pursuit of such compounds, we conducted an in vitro screening to identify agents capable of mitigating lung inflammation. Initially, we assessed the viability of human lung epithelial cells (L132) under various concentrations and exposure durations to V2O5. We observed a trend toward reduced viability at higher V2O5 concentrations after 24 h, with a pronounced dose-dependent decline after 72 h (Figure 1A). Subsequently, upon treating the cells with 10 μM V2O5, we evaluated several drugs and natural compounds for their potential anti-inflammatory effects on L132 cells. Notably, silibinin treatment restored cell viability to levels comparable to the untreated control group after 96 h (Figure 1B).

Figure 1.
Figure 1.

Silibinin effectively increased cell viability in V2O5-treated L132 cells. (A) The cells (1×103 cells/well) were treated with V2O5 at various concentrations (1, 2, 5, 10, and 15 μM of V2O5). (B) The cells were treated with 6-shogaol, silibinin, gefitinib, and imatinib with 10 μM of V2O5. Cell viability was measured by CCK-8 assay. The cell viability is expressed as an OD value (mean±SD). #p<0.05, ##p<0.01, and ###p<0.005 as compared with the control group. *p<0.05, **p<0.01, and ***p<0.005 as compared with the V2O5 group.

Silibinin attenuates V2O5-induced lung inflammation. Inflammatory responses in lung tissues are characterized by increased infiltration of inflammatory cells (24). To assess this, we conducted histological analyses using H&E staining on lung tissues collected from each experimental group. The group exposed to V2O5 alone demonstrated significant infiltration of inflammatory cells within the bronchoalveolar regions. In contrast, the group treated with silibinin in addition to V2O5 exposure showed a substantial reduction in inflammatory cell infiltration (Figure 2A). Measurements of bronchial wall thickness further corroborated these findings, with the V2O5 group exhibiting an increase in wall thickness, which was notably reduced in the silibinin 50/100 group (Figure 2B). The H score, a quantitative measure of histological inflammation, was significantly elevated in the V2O5 group relative to the control group; however, it was markedly decreased in the silibinin 50/100 group when compared to the V2O5 group (Figure 2C).

Figure 2.
Figure 2.

Effects of silibinin on inflammatory cell infiltration and bronchoalveolar inflammation in lung tissues. The control group was treated with saline only; V2O5 mice were treated with V2O5 by whole-body inhalation; Silibinin 50 and 100 mice were treated with silibinin (50 or 100 mg/kg) and V2O5 whole-body inhalation. (A) Lung tissues were stained with hematoxylin and eosin (×100; scale bar, 75 μm). The black arrows indicate epithelial cells in the lung tissue. (B) The thickness of the bronchoalveolar walls in the lung tissues was calculated as the average of measurements obtained three times in four directions. (C) The inflammatory score was calculated as an average of the response values. Data are shown as the mean±SD (n=6). #p<0.05, ##p<0.01, and ###p<0.005 as compared with the control group. *p<0.05, **p<0.01, and ***p<0.005 as compared with the V2O5 group.

Silibinin decreases the V2O5-induced increase in neutrophils and WBC in whole blood. An increase in WBCs is a common indicator of an inflammatory response, mobilizing to combat pathogens and trigger immune reactions (35). In our study, we analyzed whole blood from mice with V2O5-induced inflammation to quantify WBC counts. The V2O5 group exhibited a significant rise in WBCs, including neutrophils, lymphocytes, and eosinophils, compared to the control. Remarkably, the group treated with 50 mg/kg of silibinin demonstrated a substantial reduction in these inflammatory cell counts. While the 100 mg/kg silibinin treatment also resulted in a decrease in inflammatory cells, the reduction was less pronounced than that observed with the 50 mg/kg dosage (Figure 3A-D).

Figure 3.
Figure 3.

Silibinin effectively reduces the number of inflammatory cells in the blood. Whole blood was collected directly from the abdominal aorta post-euthanasia, and both total and differential cell counts were determined using the ADVIA 120 Hematology System. White blood cells were classified as (A) total cells, (B) neutrophils, (C) lymphocytes, and (D) eosinophils. Data are shown as the mean±SD (n=6). Data are shown as the mean±SD (n=6). #p<0.05, ##p<0.01, and ###p<0.005 as compared with the control group. *p<0.05, **p<0.01, and ***p<0.005 as compared with the V2O5 group.

Silibinin reduces inflammatory cytokine levels in mice with V2O5-induced lung injury. To quantify the expression of inflammatory cytokines, we employed real-time PCR. Typically, these cytokines are secreted following the activation of upstream signaling pathways during an inflammatory response (25, 26). We focused on the mRNA expression levels of TNF-α, IL-6, and IL-1β, which are established markers of inflammation. In the V2O5 group, we observed a significant up-regulation of these cytokines. Conversely, silibinin administration resulted in a notable down-regulation of these cytokine levels, demonstrating a dose-responsive effect (Figure 4A-C).

Figure 4.
Figure 4.

Effects of silibinin on the expression of pro-inflammatory cytokine mRNAs in lung tissue. Total RNA was isolated form lung tissues. (A) TNF-α, (B) IL-6, and (C) IL-1β mRNA were measured by real-time PCR The relative levels of mRNA were calculated based on β-actin mRNA levels. Data are shown as the mean±SD (n=6). #p<0.05, ##p<0.01, and ###p<0.005 as compared with the control group. *p<0.05, **p<0.01, and ***p<0.005 as compared with the V2O5 group.

Effects of silibinin on TNF-α and IL-1β protein levels. Western blot analysis was utilized to corroborate the real-time PCR findings regarding TNF-α and IL-1β proteins. Mice subjected to V2O5 treatment exhibited a significant up-regulation in the expression of TNF-α and IL-1β compared to the control group. In contrast, the administration of 50 mg/kg of silibinin resulted in a decrease in the expression of these proteins compared to the V2O5 group. Intriguingly, the administration of 100 mg/kg of silibinin did not demonstrate a decrease in protein expression levels, diverging from the pattern observed at the RNA level (Figure 5A-C).

Figure 5.
Figure 5.

Silibinin effectively reduced protein expression levels of inflammatory cytokines in lung tissue. Control mice treated with saline only; V2O5, mice treated with V2O5 whole-body inhalation; Silibinin 50 and 100, mice treated with silibinin (50 and 100 mg/kg) + V2O5 whole-body inhalation (A–C). The relative levels of protein expression were calculated based on β-actin protein expression levels in lung tissue. Data are shown as mean±SD (n=6). #p<0.05, ##p<0.01, and ###p<0.005 as compared with the control group. *p<0.05, **p<0.01, and ***p<0.005 as compared with the V2O5 group.

Silibinin modulates TLR4, NLRP3, NF-Embedded ImageB, and MAPK pathway activation in V2O5-induced lung inflammation. Our investigations revealed an up-regulation in pro-inflammatory cytokine expression at the protein level following V2O5 treatment. This up-regulation was mediated through key inflammatory pathways, specifically TLR4, MAPK, NF-Embedded ImageB, and NLRP3. In the V2O5 group, there was a significant increase in the activation of TLR4 and NLRP3, alongside a notable rise in pp65, a crucial mediator in the inflammatory response. Treatment with 50 mg/kg of silibinin led to a reduction in the expression of TLR4, NLRP3, and pp65, as compared to the V2O5 group (Figure 6A). Additionally, the V2O5-induced up-regulation of pERK, pJNK, and p38 was also mitigated by the 50 mg/kg silibinin treatment (Figure 6B).

Figure 6.
Figure 6.

Silibinin effectively reduced the protein expression of MAPK signaling pathway components in lung tissue. Control mice treated with saline only; V2O5, mice treated with V2O5 whole-body inhalation; Silibinin 50 and 100, mice treated with silibinin (50 and 100 mg/kg) + V2O5 whole-body inhalation. Protein expression levels of TLR4, NLRP3, and NF-Embedded ImageB signal members, pp65 and p65 (A). Protein expression levels of MAPK signal pathways (pp38, p38, pJNK, JNK, pERK, and ERK) (B).

Interestingly, while the RNA expression levels of TNF-α and IL-1β were elevated in the V2O5 group, they were significantly reduced to control levels upon treatment with both 50 and 100 mg/kg of silibinin. However, at the protein level, only the 50 mg/kg silibinin treatment led to a decrease, with the 100 mg/kg treatment not showing a similar reduction. In contrast, for the key proteins in the NF-Embedded ImageB and MAPK pathways (NLRP3, p65, p38, JNK, ERK), both the 50 and 100 mg/kg silibinin treatments resulted in a similar decrease in expression levels. Notably, TLR4 expression was reduced only with the 50 mg/kg treatment, while the 100 mg/kg treatment maintained levels comparable to those of the V2O5 group.

These findings suggest a complex and nuanced interaction between silibinin dosage and its impact on different components of the inflammatory pathways, highlighting a differential dose-response relationship in the context of V2O5-induced lung inflammation.

Effects of silibinin on the expression of NLRP3 in lung tissue. In our study, we focused on the NLRP3 inflammasome, a critical component in the inflammatory process known to induce the expression of inflammatory cytokines such as IL-1β. Anticipating that silibinin would mitigate inflammation by modulating this pathway, we assessed the expression level of NLRP3 in lung tissue through IHC staining. The results showed a marked increase in NLRP3 expression in the lung tissue of V2O5-treated mice compared to the control group. Intriguingly, silibinin treatment led to a dose-dependent decrease in NLRP3 expression when compared to the V2O5-treated mice (Figure 7). These findings indicate that silibinin effectively alleviates lung inflammation by significantly reducing NLRP3 expression in lung tissue, underscoring its potential as a therapeutic agent in inflammatory conditions.

Figure 7.
Figure 7.

Effect of silibinin treatment on NLRP3 expression in lung tissue. Lung tissues were used for immunohistochemistry using an anti-NLRP3 antibody (×100; scale bar, 75 μm). Control mice treated with saline only; V2O5, mice treated with V2O5 whole-body inhalation; Silibinin 50 and 100, mice treated with silibinin (50 and 100 mg/kg) + V2O5 whole-body inhalation. The black arrows indicate where NLRP3 is highly distributed.

Discussion

Air pollution, a significant global concern, is exacerbated by the increase in PM. Among its various components, V2O5 is particularly notable for its detrimental effects on multiple organs, especially inducing inflammatory responses in the lungs (19). Our study utilized a V2O5-induced lung injury model in mice to assess the therapeutic potential of silibinin. We discovered that silibinin effectively mitigates pulmonary inflammation by attenuating the activities of key signaling pathways, namely TLR4/MAPK/NF-Embedded ImageB, and the NLRP3 inflammasome, in vivo. This research not only underscores the toxicity associated with whole-body exposure to PM constituents but also provides a valuable model for exploring the preventive and therapeutic applications of natural compounds.

PM, a complex mixture of chemicals, contains various constituents, such as sulfates, nitrates, and metals (45, 46). Due to its minuscule size, PM can penetrate deep into the body through lung vessels, leading to multiple organ injuries, including those to the lung, brain, heart, and small intestine (47). V2O5, a toxic trace metal in PM2.5 predominantly emitted by diesel engines (48), similarly invades various organs through lung alveoli, detrimentally impacting overall health, including the reproductive, neuronal, cardiovascular, and pulmonary systems (19, 49-51). Our previous studies have shown that inhalation of vanadium oxide negatively impacts sperm motility and function through abnormal protein kinase A activity and tyrosine phosphorylation (49). Therefore, managing V2O5 in the atmosphere is crucial, as its inhalation adversely affects not just the lungs but all organs. This study highlights silibinin’s effectiveness in treating V2O5-induced lung injury, with further research needed to explore its therapeutic potential for other organ injuries caused by V2O5 exposure.

In vitro studies indicate that silibinin inhibits the inflammatory response elicited by V2O5. Among various compounds tested, silibinin was particularly effective in mitigating V2O5-induced damage in lung epithelial cells. The therapeutic benefits of silibinin were further corroborated in a mouse model of V2O5-induced lung inflammation, where it significantly reduced the infiltration of inflammatory cells. This suggests its potential in alleviating lung injury. Subsequent analysis of whole blood revealed alterations in WBC count, a sensitive marker of inflammation. Administration of silibinin led to a significant decrease in WBC count in the lung injury model, with a dosage of 50 mg/kg being more efficacious than 100 mg/kg. This finding implies that silibinin can effectively reduce WBC count without necessitating high concentrations. The dosages of 50 mg/kg and 100 mg/kg were selected based on prior studies (52-54), with 50 mg/kg being the lowest effective concentration reported. Our study confirms that 50 mg/kg is the optimal concentration for anti-inflammatory effects in V2O5-induced lung injury, and future research will explore the effects of silibinin at lower doses.

We also measured the expression of inflammatory cytokines using real-time PCR to understand the molecular changes induced by silibinin. The results revealed a significant reduction in the mRNA levels of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β. Furthermore, the protein expression of TNF-α and IL-1β, key pro-inflammatory cytokines, was significantly reduced by silibinin at a concentration of 50 mg/kg.

Previous research has highlighted various signaling pathways implicated in PM-induced inflammation (24, 55), notably the MAPK and NF-Embedded ImageB pathways (28, 56-58). Our study adds to this body of knowledge by demonstrating that silibinin effectively down-regulates these pathways, particularly TLR4, which is pivotal in initiating both chronic and acute inflammatory responses (59). Furthermore, the inhibition of the NLRP3 inflammasome by silibinin, as evidenced in our study, underscores its potential as a significant therapeutic agent in combating inflammation (60). These findings not only reinforce the detrimental impact of air pollutants like V2O5 but also position silibinin as a promising candidate in the development of treatments for pollution-related health issues. Given the global challenge posed by air pollution, the implications of our study are far-reaching, suggesting a new avenue for mitigating the health risks associated with environmental contaminants.

Conclusion

Our study demonstrates that silibinin effectively down-regulates the TLR4/MAPK/NF-Embedded ImageB/NLRP3 signaling pathways in V2O5-induced lung inflammation, suggesting a pivotal role in the regulation of inflammation. These findings are particularly significant in the context of increasing global air pollution and its associated health risks. This research not only contributes to our understanding of the molecular mechanisms underlying PM-induced inflammation but also positions silibinin as a promising therapeutic agent. Future studies should explore the broader applicability of silibinin in treating other PM-related health conditions and its potential clinical use. While our results are promising, we recommend a cautious interpretation and suggest further research to confirm these findings and fully understand the therapeutic potential of silibinin in air pollution-related diseases.

Footnotes

  • Authors’ Contributions

    MO K and SJP designed and supervised the experiment. HBI and EK designed and performed the experiment. HJK, HK, and JK analyzed and interpreted the data. HBI, EK, and YS wrote the manuscript. SHK, EJL, WSK, JY, and ZYR reviewed the manuscript. All Authors reviewed and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Funding

    This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2022R1A2C1008660).

  • Received May 27, 2024.
  • Revision received June 24, 2024.
  • Accepted June 26, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Silibinin Mitigates Vanadium-induced Lung Injury via the TLR4/MAPK/NF-κB Pathway in Mice
HOBIN IM, EUNGYUNG KIM, HONG JU KWON, HYEONJIN KIM, JIWON KO, YONGHUN SUNG, SUNG-HYUN KIM, EUN JUNG LEE, WOO-SUNG KWON, ZAE YOUNG RYOO, JUNKOO YI, SI JUN PARK, MYOUNG OK KIM
In Vivo Sep 2024, 38 (5) 2179-2189; DOI: 10.21873/invivo.13681
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