Protective effects of Silibinin and cinnamic acid against paraquat-induced lung toxicity in rats: impact on oxidative stress, PI3K/AKT pathway and miR-193a signaling

Protective effects of Silibinin and cinnamic acid against paraquat-induced lung toxicity in rats: impact on oxidative stress, PI3K/AKT pathway and miR-193a signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective effects of Silibinin and cinnamic acid against paraquat-induced lung toxicity in rats: impact on oxidative stress, PI3K/AKT pathway and miR-193a signaling Basma M. Fouad, A. A. Abdel-Ghany, Mohamed A. Kandeil, Ibrahim T. Ibrahim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4086459/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Oct, 2024 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 8 You are reading this latest preprint version Abstract Purpose Levels of reactive oxygen species (ROS) are the primary determinants of pulmonary fibrosis. It was discovered that antioxidants can ameliorate pulmonary fibrosis caused by prolonged paraquat (PQ) exposure. However, research into the precise mechanisms by which antioxidants influence the signaling pathways implicated in pulmonary fibrosis induced by paraquat is still insufficient. This research utilized a rat model of pulmonary fibrosis induced by PQ to examine the impacts of Silibinin (Sil) and cinnamic acid (CA) on pulmonary fibrosis, with a specific focus on pro-fibrotic signalling pathways and ROS-related autophagy. Methods Lung injury induced by paraquat was demonstrated to be associated with oxidative stress and inflammation of the lungs, downregulated (miR-193a) and upregulated PI3K/AKT/mTOR signaling lung tissues. Expression levels of miR-193a were determined with quantitative real-time PCR, protein level of protein kinase B (Akt), phosphoinositide 3-Kinase (PI3K) were determined by western blot analysis. Hydroxyproline levels (HYP) and Transforming growth factor-β1 (TGF-β1) were measured by ELISA, malondialdehyde(MDA), total antioxidant capacity (TAO), glutathione peroxidase (GSH) and catalase and were measured in lung tissue homogenates colorimetrically using spectrophotometer. Results Long-term exposure to paraquat resulted in decreased PI3K/AKT signalling, decreased cell autophagy, increased oxidative stress, increased and helped pulmonary fibrosis formation. Silibinin and cinnamic acid also decreased oxidative stress by increasing autophagy and miR-193a expression, which in turn decreased pulmonary fibrosis. These effects were associated by low TGF- β1. Conclusions Silibinin and cinnamic acid inhibited PQ-induced PI3K/AKT by stimulating miR-193-a expression, thus attenuating PQ- induced pulmonary fibrosis. Paraquat Silibinin cinnamic acid miRNA 193a PI3K AKT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Paraquat (N, N′- dimethyl-4,4 ′ - bipyridinium dichloride) is a quaternary nitrogen-containing compound and is a broad-spectrum herbicide that has increased agricultural production due to the variety of crops in which it is used. Other popular trade names for paraquat include methyl viologen, and paraquat dichloride (Dinis-Oliveira et al., 2008 , Tsai, 2013 ). In more than 120 countries around the world, over 100 crops are treated with paraquat, the third most popular herbicide due to its low cost.(Dinis-Oliveira et al., 2008 ) Paraquat toxicity has resulted in many cases of human poisoning and deaths due to multiple organ failure, including the nervous system, the heart, liver, and the kidneys (Wright et al., 1997 ). Remarkably, paraquat accumulates mainly in lung tissue; as a result, PQ ingestion, whether accidental or intentional, may result in death due to progressive lung fibrosis and respiratory failure(Yan et al., 2017 , Zeinvand-Lorestani et al., 2018 ). Although the guidelines for the treatment of PQ poisoning patients have not yet been thoroughly established, current therapies include supportive care alone as well as combinations of hemodialysis, immunological modulation, antioxidant therapy, and hemoperfusion (Qian et al., 2014 ). Despite these treatments, the current mortality rate of PQ poisoning is still over 50%(Xu et al., 2015 ). Oxidative stress is a crucial molecular mechanism of paraquat induced lung fibrosis, it often occurs in the lung after exposure to paraquat and alters the usual balance between oxides and peroxides while increasing the quantities of reactive oxygen species (ROS)(Kinnula and Crapo, 2003 , Faner et al., 2012 ) ,such as superoxide anion (O2−), singlet oxygen (O) as well as hydroxyl and peroxyl radicals (Suntres, 2002 ) ROS cause lung fibrosis by promoting lung cell death and reducing autophagy levels in alveolar epithelial cells.(Jones, 2006 ) The most traditional and commonly used medicinal herb is Milk Thistle (Silybum marianum), which is used for its antioxidant properties. (Negi et al., 2008 )Silymarin is derived from the plant's seeds and fruits and it was found to be a potent antioxidant.(Comar and Kirby, 2005 ) Silibinin has also been shown to have powerful anti-proliferative effects against a variety of cancer cell lines(Gazak et al., 2007 ). Furthermore, a study of the cancer chemo-preventive and anticarcinogenic properties of silymarin and Silibinin found that the effects of silymarin are related to its majot component Silibinin.(Hogan et al., 2007 ). Some studies have also proved that Silibinin has hepatoprotective effect against oxidative stress and ROS.(Haddad et al., 2011 ) Cinnamon is a plant with many uses as a herbal remedy. Its constituents include, tannin, mucilage, resin, sugar, and essential oil, the most important of which is CD, which has anti-inflammatory, antibacterial, and antioxidant qualities.(Barceloux, 2009 ) Furthermore, Cinnamic acid, a primary active phenolic component in cinnamon, has a diversity of pharmacological activities, including antioxidant and antibacterial activity (Chen et al., 2011 ). CA has strong antioxidant activity due to the existence of vinyl fragments in its structure. This property has sparked our interest in studying this natural compound as a potential target for the treatment of pathological diseases caused by oxidative stress(Babaeenezhad et al., 2021 ). The AKT/mTOR signalling pathway is a key regulator of cell growth, proliferation and survival. It is triggered by a variety of extracellular signals such as growth factors, cytokines, and hormones. The pathway consists of two main components: phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) .(Shiau et al., 2022 ) The PI3K/Akt/mTOR signalling pathway is involved in the regulation of oxidative stress, which occurs when there is an imbalance between the cell's ability to scavenge reactive oxygen species (ROS) and their production. Oxidative stress can be regulated via the PI3K/Akt/mTOR pathway, which modulates the expression of catalase and superoxide dismutase (SOD), two antioxidant enzymes. Additionally, mTOR can regulate oxidative stress by modulating mitochondrial function and autophagy(Shiau et al., 2022 ). The vital functions of microRNAs (miRNAs), an emerging class of gene expression regulators, in numerous biological processes, including cell proliferation, apoptosis, cell cycle progression and organ development, are well established (Muluhngwi and Klinge, 2015 ). Also, miR193a was found to be methylated in patients with non- small cell lung cancer. An additional investigation revealed that miR193a-3p promoted apoptosis in lung cancer cells by adversely affecting ERBB4, a protein that is frequently aberrated in human lung cancer, thereby inhibiting cell invasion and proliferation.(Liang et al., 2015 ) The purpose of this research is to determine whether cinnamic acid and silibinin can protect against paraquat-induced pulmonary toxicity via the PI3K/Akt/mTOR pathway and miRNA 193-a signalling pathway. Materials and methods 3.1 Materials 3.1.1. Animals In this investigation, male adult Wistar albino rats weighing 200–250 g have been used A clean plastic cages used for keeping them, maintaining a temperature regulation of 23 to 25°C and a (light and dark) cycle of 12 hours. Water and standard diet pellets were provided to them. To reduce physiological reactions to handling, rats were given a week to acclimatize. 3.1.2. Chemicals Silibinin, Cinnamic acid and Paraquat were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). The remaining reagents and compounds were all of analytical grade. 4.1. Methods: 4.1.2. Experimental design Six groups, each consisting of ten male Wistar albino rats, were distributed at random. For seven consecutive days, rats in the normal control group received vehicle saline and CMC (both intraperitoneal and orally). On the 7th day, the second group of rats received 0.9% saline (i.p) for 7 consecutive days with a one single dose of paraquat (30 mg/kg, intraperitoneal) (Ahmed et al., 2019 ) The 3rd group received cinnamic (50 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days with a one single dose of paraquat (30 mg/kg, intraperitoneal) on the 7th day. The 4th group received Silibinin (200 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days with a one single dose of paraquat (30 mg/kg, intraperitoneal) on the 7th day.(Lu et al., 2009 , Song et al., 2017 ) The 5th group received cinnamic acid (50 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days.(Abd El-Raouf et al., 2015 ) The 6th group received Silibinin (200 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days. 4.1.3. Collection of samples All rats were sacrificed by being decapitated 48 hours after receiving a paraquat injection. Immediately after isolation, the lungs were weighed. As soon as they were ready to be processed, all samples were kept at -80°C. 4.1.4. Histopathological examination Immediately after each animal, the upper left lung lobe was fixated in 10% neutral buffered formalin. The lungs underwent a process of progressive dehydration, paraffin embedding, sectioning into 4µm, and subsequent staining with hematoxylin and eosin (H&E). An assessment was conducted on lung specimens to identify histological changes that are indicative of ALI. These changes included interstitial edema, neutrophil infiltration, alveolar congestion, and hemorrhage. A pathologist conducting the histopathological analysis was not informed of the experimental design employed in this investigation. 4.1.5. Preparation of lung tissue homogenates Using a variable-speed homogenizer (tissue homogenizer yellow line D118 basic, Germany), the right lung tissues of the rats were immediately extracted and promptly homogenized in biphosphate buffer (pH 7.4) at concentration 0.09% at 4°C. by centrifuging lung homogenates (bench top cooling centrifuge model 2-16KL, Germany) for 15 min at 1000 g, 4°C, supernatants were obtained to be used in assessing oxidative stress-related parameters and hydroxyproline. 4.1.6. Determination of hydroxyproline in lung tissue homogenate by ELISA Through the measurement of hydroxyproline levels, the collagen content of lung tissue can be determined. The presence of collagen deposition in the lungs is suggestive of lung fibrosis. The CUSABIO kit catalog number used for carrying out the assay was CSB-E08838r. The competitive inhibition of enzyme immunoassay method is used in this assay. An antibody that is specific to Hyp has been pre-coated onto the microtiter plate that is included in this kit. Standards or samples are put into the corresponding wells of the microtiter plate using Hyp-conjugated Horseradish Peroxidase (HRP). The competitive inhibition reaction is initiated when Hyp conjugated with HRP is combined with Hyp in the samples. The color that results from adding a substrate solution to the wells is inversely proportional to the quantity of Hyp present in the sample. The process of color development stops, and the color's intensity is assessed. 4.1.7. Determination of oxidative stress biomarkers in lung homogenate: The activity of MDA and TAC is determined by inhibiting chromagen reduction; this was accomplished using colorimetric assays obtained from Biodiagnostic (cat. no. TA 25 13., CAT. no. MD 25 29). The MDA assay reagent operates by utilizing the reaction between MDA and thiobarbituric acid in the sample to produce an MDA-TBA adduct. This adduct is subsequently quantified colorimetrically at an absorbance of 532 nm. The TAC assay, conversely, relies on the conversion of Cu2 + to Cu + via the action of small molecule antioxidants, such as GSH. A colorimetric probe was utilized to chelate the reduced Cu + ion, resulting in an absorbance peak at 570 nm OD that is directly proportional to the overall antioxidant capacity. Using reagent CAT. No. GP 2524, reduced glutathione (GSH) levels in lung tissue homogenates were determined. The assay measures the activity of c-GPx in an indirect fashion. The reduction of an organic peroxide by c-GPx generates oxidized glutathione (GSSG), which is subsequently recycled back to its reduced state via the enzyme glutathione reductase (GR). The colorimetric assay for catalase is performed using biodiagnostic kit CAT. No. CA 25 − 17.Its fundamental principle is that catalase must react with a predetermined amount of H2O2. The reaction stops precisely one minute later when a catalase inhibitor is applied. When peroxidase (HRP) is present, the remainder of H2O2 undergoes a reaction with 3,5-dichloro-2-hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone (AAP). This reaction produces a chromophore whose wavelength of 510 nm is inversely proportional to the quantity of catalase present in the initial sample. 4.1.8. Determination of TGF-β1 using ELISA This assay was performed using the ER1378 FineTest biotechnology reagent (Wuhan, China). The sandwich enzyme-linked immune-sorbent assay technology formed the core of this reagent. The capture antibody was coated to 96-well plates via pre-coating. Additionally, detection antibodies were biotin-conjugated antibodies. Following the addition of the standards, test samples, and biotin-conjugated detection antibody to the wells, and to remove the mixture a wash buffer was used. After adding HRP-Streptavidin, any unbound conjugates were removed by rinsing with wash buffer. By using TMB substrates, the HRP enzymatic reaction could be observed. TMB was removed from its blue form by HRP catalysis, which was reversed to yellow upon addition of an acidic solution to stop reaction. The density of yellow color is proportional to the quantity of the target sample that is captured on the plate. Using a microplate reader to measure the O.D. absorbance at 450nm enables one to calculate the concentration of the target. 4.Determination of phosphatidyl inositol-3-kinase (PI3K) and protein kinase B(Akt) protein expression in the lung tissue of rats by western blot analysis Lung tissue homogenate was centrifuged in accordance with a procedure already described. The supernatant was subsequently collected, and the concentration of total protein was ascertained using the Micro BCA protein assay reagent (Cat. No: ARG66216; Arigo Biolaboratories; Rabbit pAb). Following electrophoresis on 8% polyacrylamide gel, protein samples were transferred to nitrocellulose membranes.Non-specific binding sites were blocked by incubating in TBST (0.05% Tween 20 in Tris buffered saline) and 7.5% (w/v) non-fat powdered milk for two hours at room temperature. After that, membranes were cleansed with TBST for ten minutes. Subsequently, the primary specific antibodies targeting PI3K and Akt were diluted at a 1:1000 ratio and incubated at 4°C overnight (Cell Signaling Technology, USA). Following membrane washing, the secondary antibody is (horseradish peroxidase-conjugated anti-rabbit IgG antibody) ,it was applied at a 1:25000 dilution (Bio-Rad, USA) and incubated at room temperature for one hour. This was subsequently followed by further rinsing. Using an enhanced chemiluminescence ECL Plus System (Amersham Biosciences, USA), immunocomplexes were observed. Applying densitometry and Molecular Analyst Software (Bio-Rad, USA), the quantities were determined. The expression of PI3K and proteins was quantified in relation to β-actin. 4.1.10. Determination of miRNA 193-a using qRT-PCR To quantify the expression of miR193a, microRNAs were isolated using the All-in-One™ miRNA qRT-PCR Detection Kit 2.0 (Gene Copeia TM, Rockville, USA) Cat. No. QP115 (20 RT and 200 qPCR reactions) and Cat. No. QP116 (60 RT and 600 qPCR reactions). To analyze miRNA, cDNAs containing transcribed miRNAs were amplified via PCR using the All-in-One miRNA qPCR Primer (miR-193a). Following purification with TRIzol (Invitrogen), miRNAs were transcribed into cDNAs. The primers for PCR, which are detailed in Table 2 , were subsequently utilized. For qRT-PCR, a Fast Real-Time PCR System from Applied Biosystems 7900 (Foster City, CA, USA) was utilized. As the endogenous control gene, U6 (Universal Adaptor PCR Primer) was used to normalize the input quantities. To normalize expression levels, endogenous controls were used. The determination of fold variations in expression levels was performed utilizing the 2 − ΔΔCt method. The gene sequences of the PCR primers is shown in Table 1 . Table 1 effect of cinnamic acid and Silibinin on paraquat induced lung toxicity in rats by measuring miRNA-193a-3p using qRT-PCR. Primers Forward Reverse miR-193a-3p 5′-GTT TGG TAG CTT ATC AGA CTG A-3′ 5′-GTG CAG GGT CCG AGG T-3′ U6 (Internal control) 5′-CTC GCT TCG GCA GCA CA-3′ 5′-AAC GCT TCA CGA ATT TGC GT-3′ Table 2 The Effect of paraquat, Silibinin and cinnamic acid and their combination on oxidative stress markers in the lung tissue of rats. parameter Normal Control PQ CA + PQ Silibinin + PQ CA Silibinin CAT (U/gT) 2.9033 ± 0.08 1.0414 ± 0.09 a 1.8067 ± 0.11 b 2.15 ± 0.07 b 2.835 ± 0.08 2.812 ± 0.09 TAC (mM/L) 1.9017 ± 0.05 0.69 ± 0.05 a 1.1944 ± 0.07 b 1.415 ± 0.03 b 1.843 ± 0.09 1.846 ± 0.06 MDA (nmol/gT) 0.9033 ± 0.06 1.8743 ± 0.08 a 1.5533 ± 0.10 b 1.3483 ± 0.02 b 0.963 ± 0.05 0.97 ± 0.04 GPx (U/gT) 2.215 ± 0.06 0.7929 ± 0.06 a 1.3767 ± 0.09 b 1.645 ± 0.04 b 2.108 ± 0.12 2.126 ± 0.14 HYP (ng/ml) 0.143 ± 0.03 0.532 ± 0.03 a 0.39 ± 0.05 b 0.265 ± 0.02 b 0.161 ± 0.01 0.162 ± 0.02 Paraquat (30 mg/kg, i.p.,one single dose), Cinnamic acid (50 mg/kg, oral ), Silibinin ( 200 mg/kg oral). Data were expressed as mean ± SD, (n = 10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. (a) differs significantly from the normal control group (p < 0.001), and (b) differs significantly from the paraquat group (p < 0.001). Statistical analysis Data were analyzed statistically by SPSS (statistical package for social sciences), version 28, IBM software (NY, USA). The data were expressed as mean ± SD (standard deviation). SPSS was utilized to conduct one-way ANOVAs followed by Tukey post hoc tests for statistical comparisons between groups; p-values less than or equal to 0.001 considered to be significant. Results 6.1. Histopathological examination result: As shown in (Fig. 1 ) A: Normal lung tissue, the alveolar spaces were patent and lined by normal pneumocytes (black arrows) (H&E X 200) B: Diseased lung tissue, the alveolar septa were markedly edematous, congested, infiltrated by chronic inflammatory cells (black arrow), peribronchial inflammations and epithelial desquamation (red arrow) (H&E X 200) C: The alveolar spaces were patent and the alveolar sepata retain to the normal state with minimal residual inflammations (black arrows) (H&E X 100) (excellent response to toxicity). D: The alveolar septa were mildly edematous, congested and infiltrated by chronic inflammatory cells (black star) (H&E X 100) (moderate response to toxicity) 6.2 Effect of paraquat, Silibinin, cinnamic acid and their combination on oxidative stress markers in lung tissue homogenates in rats: As displayed in (Table 2 ) and( Fig. 2 A), CAT (catalase) level in the PQ group is significantly increased in comparison to the normal control group by 64.13% (p < 0.001), on the other hand the CA and Sil administration to PQ-treated rats significantly reduced CAT level by 73.4% (p < 0.001) and 106.6% (p < 0.001) respectively in comparison to PQ group. HYP the hydroxyproline level significantly increased in PQ group in comparison to normal control group by 271.7% (p < 0.001). on the other hand, the CA and Sil administration to paraquat treated rats significantly decreased HYP level in comparison to paraquat group by 26.77% (p < 0.001) and 50.19% (p < 0.001) respectively, (Fig. 2 B). GPx (glutathione peroxidase) level in the PQ group is significantly decreased as compared to the normal control group by 64.21% (p < 0.001), on the other hand the CA and Sil administration to paraquat treated rats significantly increased GPx level as compared to the PQ group by 73.7% (p < 0.001) and 107.5% (p < 0.001) respectively, (Fig. 2 C) MDA (malondialdehyde) level in the PQ group is significantly increased as compared to the normal control group by 107.3% (p < 0.001), on the other hand the CA and Sil administration to paraquat treated rats significantly decreased the MDA level as compared to the PQ group by 17.11% (p < 0.001) and 28.1% (p < 0.001) respectively, (Fig. 2 D). TAC (total antioxidant capacity) level in the PQ group decreases significantly in comparison to the normal control group by 63.74% (p < 0.001), on the other hand the CA and Sil administration to paraquat treated rats significantly increased the TAC level in comparison to the PQ group by 73.1% (p < 0.001) and 105.3% (p < 0.001) respectively, (Fig. 2 E) 6.3 Effect of paraquat, Silibinin, cinnamic acid and their combination on TGF-β1 in lung tissue homogenates in rats using ELISA : As we observe in (Fig. 3 ), paraquat significantly increased TGF-β1 level in the lung tissue homogenates of rats by 113.6% p < 0.001 in comparison to normal control group. On the other hand, CA and Sil administration to PQ- treated rats decrease significantly the TGF-β1 level by 15.8% P < 0.001 and 26.3% P < 0.001. 6.3 Effect of paraquat, Silibinin, cinnamic acid and their combination on AKT and PI3K in lung tissue homogenates in rats using western blot analysis : As illustrated in (Fig. 4 and Fig. 5 ), The expression of the PI3K and AKT proteins in the lung tissue of rats administered Paraquat increased significantly by 158.7% p < 0.001 and 193% p < 0.001, respectively, in comparison to the normal control group. On the other hand, the administration of cinnamic acid and Silibinin to PQ- treated rats showed a significant decrease in PI3K protein expression in the lung tissues of rats by 22.4% p < 0.001 and 22% p < 0.001 respectively, and significant decrease in AKT protein expression in the lung tissues of rats by 26% p < 0.001 and 27.6% p < 0.001 respectively. 6.4 Effect of paraquat, Silibinin, cinnamic acid and their combination on expression of miRNA-193a-3p using qRT-PCR : As illustrated in (Figure 4 and Figure 5), The expression of the PI3K and AKT proteins in the lung tissue of rats administered Paraquat increased significantly by 158.7% p < 0.001 and 193% p < 0.001, respectively, in comparison to the normal control group. On the other hand, the administration of cinnamic acid and Silibinin to PQ- treated rats showed a significant decrease in PI3K protein expression in the lung tissues of rats by 22.4% p < 0.001 and 22% p < 0.001 respectively, and significant decrease in AKT protein expression in the lung tissues of rats by 26% p < 0.001 and 27.6% p < 0.001 respectively. 6.5 Effect of paraquat, Silibinin , cinnamic acid and their combination on expression of miRNA-193a-3p using qRT-PCR : Here in Fig. 6 , We explored the effects of Silibinin and cinnamic acid on miRNA 193-a expression levels in fibrotic tissues of lung obtained from rats received one single dose of PQ (30 mg/kg). MiRNA-193a expression levels in lung tissues were assessed with qRT-PCR. MiR-193a expression levels were decreased in the lungs during paraquat-induced lung fibrosis in comparison to the normal control group by 62% (P < 0.001). Conversely, miR-193a expression was significantly increased in cinnamic acid and Silibinin -treated rats in comparison to the Paraquat group by 64.7% (P < 0.001) and 78.5% (P < 0.001) respectively. 6.6. Correlation between miRNA 193a and other parameters: In Fig. 7 A ,there is a correlation between miRNA 193-a and transforming growth factor β1, where R= -0.96 and p < 0.001. This proves a very strong relationship mechanism between both parameters. the correlation between miRNA 193-a and serine/threonine kinase 1 (Akt) is obvious, where R= -0.94 and p < 0.001. This proves a very powerful relationship mechanism between both parameters, Fig. 7 B. And at last, the correlation between miRNA 193-a and hydroxyproline, where R= -0.94 and p < 0.001. This proves a very strong correlation mechanism between both parameters, Fig. 7 C. 6.7. A graph to summarize the Mechanism of our study. In Fig. 8 ,this graph presents and illustrates our study in brief, where it shows the effect of each of paraquat, Silibinin and cinnamic acid on lung tissue of rats and pulmonary fibrosis. Also, it shows the mechanism of PI3K/AKT pathway and its relationship with miRNA 193-a, growth factor β1 and collagen deposition in lungs. Discussion With over a hundred uses on crops in over 120 countries, paraquat is the third most widely used pesticide in the world. Severe lung damage and Parkinson's disease (PD) are the consequences of PQ poisoning. Self-induced poisoning is a significant public health problem in developing countries and is associated with PQ (Tsai, 2013 ). Since its introduction in agriculture, accidental or deliberate ingestion has resulted in thousands of deaths each year. PQ has been identified as a significant health hazard due to its potential to cause severe lung damage in both humans and experimental animals. It has been extensively studied as a pulmonary toxicant. (Tyagi and Singh, 2020 ) The prophylactic effects of cinnamic acid and Silibinin against paraquat-induced lung toxicity may be attributed to their antioxidant and anti-inflammatory properties. These compounds have been reported to scavenge free radicals, reduce oxidative stress, and inhibit the production of pro-inflammatory cytokines. Additionally, cinnamic acid and Silibinin may modulate various signalling pathways involved in oxidative stress and inflammation, such as AKT/mTOR pathway. inhibition of the PI3K/AKT pathway can enhance the expression of antioxidant enzymes and promote cellular defense against oxidative damage. Furthermore, cinnamic acid and Silibinin have been shown to possess anti-fibrotic properties, which may contribute to their protective effects against lung toxicity. These compounds can inhibit the activation of fibroblasts and the deposition of extracellular matrix components, thereby preventing tissue fibrosis. In our study, the administration of paraquat to rats resulted in the development of pulmonary toxicity, as indicated by increased levels of oxidative stress markers (hydroxyproline and lipid peroxides) in tissue homogenates. The results indicate that paraquat administration significantly decreased the levels of catalase (CAT), total antioxidant capacity (TAC), and glutathione peroxidase (GPx) in the lungs of rats. On the contrary, the paraquat group exhibited a significant elevation in malondialdehyde (MDA) and hydroxyproline concentrations when compared to the normal control group. Prior research has established that reactive oxygen species, namely hydrogen peroxide and superoxide radicals, are produced when the enzyme NADPH-Cyt-p-450 reductase metabolizes paraquat.(Yumino et al., 2002 ) The accumulation of superoxide radicals has the potential to induce lipid peroxidation, reduce the overall antioxidant capacity, and promote cytokine formation in lung tissues (Tyagi and Singh, 2020 ). However, the elevated lung hydroxyproline levels in the current study may serve as an early indicator of fibrosis, as hydroxyproline is a major component of collagen (Huang et al., 2021 ). Cinnamic acid is a main constituent of cinnamon. This compound has been reported to be effective against cisplatin toxicity in rats and also to reduce the inflammatory markers as well (El-Sayed et al., 2013 ). Silybin as well, or Silibinin, is a flavonoid that is derived from Silybum marianum, also known as Milk thistle. It has been reported that Silibinin reduces peroxidation of lipids, an indicator of oxidative lipids, in hepatocyte microsomes and isolated hepatocytes, suggesting that it may provide protection against oxidative stress (Lu et al., 2009 ). In the current study pre-treatment with Cinnamic acid and Silibinin resulted in a significant increase in CAT, TAC, and GPx levels, while MDA and hydroxyproline levels decreased significantly in comparison to the paraquat group. Presently, among all cytokines studied, the growth factor β family of proteins has shown the most significant effect on the deposition of extracellular matrix. TGF-β1, which is secreted in vitro as a latent precursor, stimulates the expression of fibroblast procollagen genes and the synthesis of proteins containing this protein (Xiangdong et al., 2011 ). TGF- β1 increased alveolar permeability of the epithelial cells in vitro through a mechanism involving intracellular glutathione depletion. Anti-TGF- β1 antibodies prevented lung injury in hemorrhaging rodents, according to another study (Shenkar et al., 1994 ). Previous studies proved that cinnamon extracts has potent antioxidant activity and also reduced the levels of TGF-β1 activity(Barceloux, 2009 ). Furthermore, a previous study proved that Silibinin promoted valsartan's anti-fibrotic effect by inhibiting the TGF-β1 signalling pathway(Liu et al., 2020 ). This is confirmed by our results, where PQ induced TGF-β-1 activity, but pre-treatment with cinnamic acid and silibinin reduced its levels in PQ-intoxicated lungs. As confirmed from previous studies, the proteins phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) are recognized for their involvement in the processes of wound healing. Stimulation of the PI3K/AKT signaling pathway is recognized as the fundamental factor in keloid pathogenesis by inducing collagen synthesis (Kim et al., 2017 ). Growth factors and cytokines stimulate tuberous sclerosis 1 proteins one and two in response to physiological stimuli by phosphorylating Akt and PI3K, which subsequently stimulates mTOR. And as previously described, paraquat contributes to the pathogenesis of pulmonary fibrosis by activating the PI3K/Akt pathway in alveolar epithelial cells and inhibiting autophagy activity (Jiang et al., 2021 , Shi et al., 2022 ), whereas silibinin has previously been shown to inactivate the PI3K/Akt pathway in kidney cancer cells (Yassin et al., 2021 ). Cinnamic acid as well inhibited the propagation of gastric cancer cells by downregulating the PI3K/Akt pathway (Li and Hu, 2020 ). In this study we confirmed this evidence where AKT and PI3K were inhibited by Silibinin and cinnamic acid in PQ-treated rats. A specific methylation pattern of miR-193a was found in patients with non-small cell lung cancer. By analysing the expression patterns of miR-193a-3p in NSCLC tissues, it was demonstrated that miR-193a-3p may act as a tumour suppressor in NSCLC and regulate the signalling of the PI3K/Akt pathway by targeting KRAS (Fan et al., 2017 , Khordadmehr et al., 2019 ). Another study found that miR-193a-3p inhibited lung cancer cell proliferation and invasion while promoting apoptosis (Liang et al., 2015 ). Paraquat was proved to have impact on the miRNA 193a expression by its inhibition and thus inducing oxidative stress in lung cells (Liu et al., 2019 ). And this is similar to what we have shown in our study, the rats treated only with PQ have lower levels of miRNA 193-a expression than those treated with cinnamic acid and silibinin. Conclusion Our study illustrated that paraquat could induce pulmonary toxicity and fibrosis by enhancement of oxidative stress and collagen deposition in lung tissues and consequently increasing level of TGF-β1, also affecting pathway of (PI3K/Akt) by inducing it and inhibiting the signaling of (miRNA 193-a) in lung tissues. Cinnamic acid and Silibinin on the other hand offered a new prophylactic effect against PQ toxicity by downregulation of (PI3K/Akt) pathway and increasing the expression level of miRNA 193-a in the lung tissues. More studies are recommended for the synergistic effect of combination cinnamic acid and Silibinin as protective agents against lung toxicity. Investigation of the pulmonary protective effects of cinnamic acid and Silibinin in patients intoxicated with paraquat is recommended through clinical trials. Declarations Acknowledgement: The authors would like to express their appreciation for EL NAHDA university animal house, Dr. NM Ahmed and Dr. Mira Magdy for their support in the statistical advisory and their help in the preparation of the manuscript. Ethical approval Faculty of Pharmacy of Benisuef University, Egypt, approved the current research in accordance with ARRIVE guidelines (Animal Research: Reporting of In-Vivo Experiments).This research was approved by BSU-IACUC reviewers and the approval number is (022-529).The National Institutes of Health's (NIH) guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and the local institutional Research Ethics Committee's approval were both followed in the carrying out of each experiment in respect to the ARRIVE guidelines (Animal Research: Reporting of In-Vivo Experiments). 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Pediatric emergency care 31: 117-121 Yan B, Chen F, Xu L, Xing J, Wang X (2017) HMGB1-TLR4-IL23-IL17A axis promotes paraquat-induced acute lung injury by mediating neutrophil infiltration in mice. Scientific reports 7: 597 Yassin NYS, AbouZid SF, El-Kalaawy AM, Ali TM, Elesawy BH, Ahmed OM (2021) Tackling of Renal Carcinogenesis in Wistar Rats by Silybum marianum Total Extract, Silymarin, and Silibinin via Modulation of Oxidative Stress, Apoptosis, Nrf2, PPARgamma, NF-kappaB, and PI3K/Akt Signaling Pathways. Oxid Med Cell Longev 2021: 7665169 Yumino K, Kawakami I, Tamura M, Hayashi T, Nakamura M (2002) Paraquat-and diquat-induced oxygen radical generation and lipid peroxidation in rat brain microsomes. The journal of biochemistry 131: 565-570 Zeinvand-Lorestani H, Nili-Ahmadabadi A, Balak F, Hasanzadeh G, Sabzevari O (2018) Protective role of thymoquinone against paraquat-induced hepatotoxicity in mice. Pesticide biochemistry and physiology 148: 16-21 Additional Declarations No competing interests reported. Supplementary Files AKTPI3Kwesternblotanalysis.png Cite Share Download PDF Status: Published Journal Publication published 25 Oct, 2024 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted Editorial decision: Revision requested 30 Jun, 2024 Reviews received at journal 25 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers agreed at journal 03 Apr, 2024 Reviewers invited by journal 03 Apr, 2024 Submission checks completed at journal 28 Mar, 2024 Editor assigned by journal 28 Mar, 2024 First submitted to journal 12 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4086459","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285059963,"identity":"7ec3eb2c-6cc2-4afc-9ff3-d508206595e0","order_by":0,"name":"Basma M. Fouad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYBACAzjrBhB/YGBIQBEkpIWxcQbJWpp5iNFizn786YYfDNvk+G43P39su+NwHgN78zYJhopanFose3LMbvYw3DaWvHPMsDn3zOFiBp5jZRIMZ47jdtiBHLYbPAy3EzfcSABqaTuc2CCRYybB2HYMt5bzz5/d/MNwu37DjfSPzZYgLfJvgFr+4dFyI8HsNtCWBIMbOYbNjGBbeIBaGmpw+2XGG7PbMga3DWfeyCmc2duWXszGk1ZskXDsAE4t5vzpz26+qbgtz3cjfcOHn23Wefzshzfe+FBTh1ML1HlIbDYQkcBwmIAWLICQLaNgFIyCUTCCAAA88V5XQm6g0AAAAABJRU5ErkJggg==","orcid":"","institution":"Nahda University","correspondingAuthor":true,"prefix":"","firstName":"Basma","middleName":"M.","lastName":"Fouad","suffix":""},{"id":285059964,"identity":"3838d24c-8257-4e5b-a7aa-973010501641","order_by":1,"name":"A. A. Abdel-Ghany","email":"","orcid":"","institution":"Nahda University","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"A.","lastName":"Abdel-Ghany","suffix":""},{"id":285059965,"identity":"fc86a14f-a0f7-4e82-b472-775b65c38122","order_by":2,"name":"Mohamed A. Kandeil","email":"","orcid":"","institution":"Beni-Suef University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"A.","lastName":"Kandeil","suffix":""},{"id":285059967,"identity":"c4f5eb35-ea8a-405d-a431-07e10f3d9060","order_by":3,"name":"Ibrahim T. Ibrahim","email":"","orcid":"","institution":"Beni-Suef University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"T.","lastName":"Ibrahim","suffix":""}],"badges":[],"createdAt":"2024-03-12 16:55:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4086459/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4086459/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-024-03511-y","type":"published","date":"2024-10-25T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53850819,"identity":"15cfa319-491a-4a06-9b15-53f4d533620e","added_by":"auto","created_at":"2024-04-01 09:56:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1524727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological examination of lung tissues treated with paraquat, Silibinin with paraquat and cinnamic with paraquat.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA): normal lung tissue (control)\u003c/p\u003e\n\u003cp\u003eB): diseased lung tissue (PQ 30 mg/kg, i.p. single dose on the 7\u003csup\u003eth\u003c/sup\u003e day)\u003c/p\u003e\n\u003cp\u003eC): pretreated rats with Silibinin. Sil 200mg/kg ,orally for 7 days) then PQ (30mg/kg ) single dose,i.p. on the 7\u003csup\u003eth\u003c/sup\u003e day.\u003c/p\u003e\n\u003cp\u003eC): pretreated rats with Cinnamic acid. CA 50 mg/kg ,orally for 7 days) then PQ (30mg/kg ) single dose,i.p. on the 7\u003csup\u003eth\u003c/sup\u003e day.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/45fe8025431309bcec2bf1fd.png"},{"id":53850817,"identity":"5daf6b96-51f6-4b3b-9c19-4e9ab0ef399e","added_by":"auto","created_at":"2024-04-01 09:56:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":222607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical illustration of the effect of paraquat, Silibinin and cinnamic acid and their combination on oxidative stress markers in the lung tissue of rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) effect of paraquat, cinnamic acid and Silibinin on catalase level in lung tissue homogenate.\u003c/p\u003e\n\u003cp\u003eB) effect of paraquat, cinnamic acid and Silibinin on hydroxyproline level in lung tissue homogenate.\u003c/p\u003e\n\u003cp\u003eC) effect of paraquat, cinnamic acid and Silibinin on glutathione perioxidase level in lung tissue homogenate.\u003c/p\u003e\n\u003cp\u003eD) effect of paraquat, cinnamic acid and Silibinin on Malonaldehyde level in lung tissue homogenate.\u003c/p\u003e\n\u003cp\u003eE) effect of paraquat, cinnamic acid and Silibinin on total antioxidant capacity level in lung tissue homogenate.\u003c/p\u003e\n\u003cp\u003eParaquat (30 mg/kg, i.p., single dose), Cinnamic acid ( 50 mg/kg, oral ), Silibinin ( 200 mg/kg oral). Data were expressed as mean ± SD, (n=10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. (a) differs significantly from the normal control group (p \u0026lt; 0.001), and (b) differs significantly from the paraquat group (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/71a70c3ec1f6d8da7fc2f3d6.png"},{"id":53850822,"identity":"1686540e-4581-4b66-9fe8-3443d6d0399e","added_by":"auto","created_at":"2024-04-01 09:56:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":89635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cinnamic acid and Silibinin on PQ- treated rats by measuring the TGF-β1 level in tissue homogenate using ELISA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaquat (30 mg/kg, i.p.,one single dose), Cinnamic acid (50 mg/kg, oral ), Silibinin ( 200 mg/kg oral ).Data were expressed as mean ± SD, (n=10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. (a) differs significantly from the normal control group (p \u0026lt; 0.001), and (b) differs significantly from the paraquat group (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/1e7a724e4474f53f672ce5c7.png"},{"id":53850821,"identity":"595e6736-6c91-40e8-87c9-6caa78011c4e","added_by":"auto","created_at":"2024-04-01 09:56:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cinnamic acid and Silibinin on PQ- treated rats by measuring phosphatidyl inositol-3-kinase PI3K using western blot.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. (4A): A representation of western blot of lung tissue PI3K protein expression.\u003c/p\u003e\n\u003cp\u003eFig. (4B): Graphical illustration of lung PI3K protein expression.\u003c/p\u003e\n\u003cp\u003eParaquat (30 mg/kg, i.p., single dose), Cinnamic acid (50 mg/kg, oral), Silibinin (200 mg/kg oral). Data were expressed as mean ± SD, (n=10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. (a) differs significantly from the normal control group (p \u0026lt; 0.001), and (b) differs significantly from the paraquat group (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/82c98436914a89e040de3a69.png"},{"id":53850824,"identity":"f009dd15-470c-44a9-97c3-8463f0f568be","added_by":"auto","created_at":"2024-04-01 09:56:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cinnamic acid and Silibinin on PQ treated rats by measuring protein kinase B (AKT) protein expression in lung tissues of rats using western blot.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. (5A): A representation of western blot of lung tissue AKT protein expression.\u003c/p\u003e\n\u003cp\u003eFig. (5B): Graphical illustration of lung Akt protein expression.\u003c/p\u003e\n\u003cp\u003eParaquat (30 mg/kg, i.p., single dose), Cinnamic acid (50 mg/kg, oral), Silibinin (200 mg/kg oral). Data were expressed as mean ± SD, (n=10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. (a) differs significantly from the normal control group (p \u0026lt; 0.001), and (b) differs significantly from the paraquat group (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/1f603c2c9f2acbfa48cc6ebb.png"},{"id":53850820,"identity":"0da5262d-f40b-4eb0-8657-0c018219ee37","added_by":"auto","created_at":"2024-04-01 09:56:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eeffect of cinnamic acid and Silibinin on PQ-treated rats by measuring miRNA 193-a gene expression by RT- PCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaquat (30 mg/kg, i.p., single dose), Cinnamic acid (50 mg/kg, oral), Silibinin (200 mg/kg oral). Data were expressed as mean ± SD, (n=10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. (a) differs significantly from the normal control group (p \u0026lt; 0.001), and (b) differs significantly from the paraquat group (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/e71294d4ef2e4836e317d8de.png"},{"id":53850825,"identity":"99293422-6a7d-49fc-b970-031311a42783","added_by":"auto","created_at":"2024-04-01 09:56:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":144719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA scattered chart showing the correlation between miRNA 193-a and TGF β1 , between miRNA 193-a and AKT and also between miRNA 193-a and Hydroxyproline.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/d5e41309883449b6f8d83390.png"},{"id":53850823,"identity":"5c63b7c2-cb16-4aa2-bc63-a63150977a2e","added_by":"auto","created_at":"2024-04-01 09:56:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":265443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIillustrated diagram for the mechanism of each of paraquat, cinnamic acid and Silibinin on lung toxicity and fibrosis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/e592d1b0b5c3da783d4986f4.png"},{"id":67681900,"identity":"0544ebd9-7c8f-49ba-be52-5a37b8dec3da","added_by":"auto","created_at":"2024-10-28 16:11:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3850360,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/44b32f09-aa42-44b8-8d26-7fb49817ae8d.pdf"},{"id":53851535,"identity":"2c9de588-86a0-4436-92ed-aae6b7103a53","added_by":"auto","created_at":"2024-04-01 10:04:23","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":45588,"visible":true,"origin":"","legend":"","description":"","filename":"AKTPI3Kwesternblotanalysis.png","url":"https://assets-eu.researchsquare.com/files/rs-4086459/v1/89b6a3d99eca2f2985be7e99.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective effects of Silibinin and cinnamic acid against paraquat-induced lung toxicity in rats: impact on oxidative stress, PI3K/AKT pathway and miR-193a signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParaquat (N, N\u0026prime;- dimethyl-4,4 \u0026prime; - bipyridinium dichloride) is a quaternary nitrogen-containing compound and is a broad-spectrum herbicide that has increased agricultural production due to the variety of crops in which it is used. Other popular trade names for paraquat include methyl viologen, and paraquat dichloride (Dinis-Oliveira et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Tsai, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In more than 120 countries around the world, over 100 crops are treated with paraquat, the third most popular herbicide due to its low cost.(Dinis-Oliveira et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eParaquat toxicity has resulted in many cases of human poisoning and deaths due to multiple organ failure, including the nervous system, the heart, liver, and the kidneys (Wright et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Remarkably, paraquat accumulates mainly in lung tissue; as a result, PQ ingestion, whether accidental or intentional, may result in death due to progressive lung fibrosis and respiratory failure(Yan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Zeinvand-Lorestani et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the guidelines for the treatment of PQ poisoning patients have not yet been thoroughly established, current therapies include supportive care alone as well as combinations of hemodialysis, immunological modulation, antioxidant therapy, and hemoperfusion (Qian et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite these treatments, the current mortality rate of PQ poisoning is still over 50%(Xu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Oxidative stress is a crucial molecular mechanism of paraquat induced lung fibrosis, it often occurs in the lung after exposure to paraquat and alters the usual balance between oxides and peroxides while increasing the quantities of reactive oxygen species (ROS)(Kinnula and Crapo, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Faner et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) ,such as superoxide anion (O2\u0026minus;), singlet oxygen (O) as well as hydroxyl and peroxyl radicals (Suntres, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) ROS cause lung fibrosis by promoting lung cell death and reducing autophagy levels in alveolar epithelial cells.(Jones, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe most traditional and commonly used medicinal herb is Milk Thistle (Silybum marianum), which is used for its antioxidant properties. (Negi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)Silymarin is derived from the plant's seeds and fruits and it was found to be a potent antioxidant.(Comar and Kirby, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eSilibinin has also been shown to have powerful anti-proliferative effects against a variety of cancer cell lines(Gazak et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Furthermore, a study of the cancer chemo-preventive and anticarcinogenic properties of silymarin and Silibinin found that the effects of silymarin are related to its majot component Silibinin.(Hogan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Some studies have also proved that Silibinin has hepatoprotective effect against oxidative stress and ROS.(Haddad et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eCinnamon is a plant with many uses as a herbal remedy. Its constituents include, tannin, mucilage, resin, sugar, and essential oil, the most important of which is CD, which has anti-inflammatory, antibacterial, and antioxidant qualities.(Barceloux, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) Furthermore, Cinnamic acid, a primary active phenolic component in cinnamon, has a diversity of pharmacological activities, including antioxidant and antibacterial activity (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). CA has strong antioxidant activity due to the existence of vinyl fragments in its structure. This property has sparked our interest in studying this natural compound as a potential target for the treatment of pathological diseases caused by oxidative stress(Babaeenezhad et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe AKT/mTOR signalling pathway is a key regulator of cell growth, proliferation and survival. It is triggered by a variety of extracellular signals such as growth factors, cytokines, and hormones. The pathway consists of two main components: phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) .(Shiau et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe PI3K/Akt/mTOR signalling pathway is involved in the regulation of oxidative stress, which occurs when there is an imbalance between the cell's ability to scavenge reactive oxygen species (ROS) and their production. Oxidative stress can be regulated via the PI3K/Akt/mTOR pathway, which modulates the expression of catalase and superoxide dismutase (SOD), two antioxidant enzymes. Additionally, mTOR can regulate oxidative stress by modulating mitochondrial function and autophagy(Shiau et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe vital functions of microRNAs (miRNAs), an emerging class of gene expression regulators, in numerous biological processes, including cell proliferation, apoptosis, cell cycle progression and organ development, are well established (Muluhngwi and Klinge, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Also, miR193a was found to be methylated in patients with non- small cell lung cancer. An additional investigation revealed that miR193a-3p promoted apoptosis in lung cancer cells by adversely affecting ERBB4, a protein that is frequently aberrated in human lung cancer, thereby inhibiting cell invasion and proliferation.(Liang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe purpose of this research is to determine whether cinnamic acid and silibinin can protect against paraquat-induced pulmonary toxicity via the PI3K/Akt/mTOR pathway and miRNA 193-a signalling pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e3.1 Materials\u003c/h2\u003e\n \u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.1.1. Animals\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eIn this investigation, male adult Wistar albino rats weighing 200\u0026ndash;250 g have been used A clean plastic cages used for keeping them, maintaining a temperature regulation of 23 to 25\u0026deg;C and a (light and dark) cycle of 12 hours. Water and standard diet pellets were provided to them. To reduce physiological reactions to handling, rats were given a week to acclimatize.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e3.1.2. Chemicals\u003c/h2\u003e\n \u003cp\u003eSilibinin, Cinnamic acid and Paraquat were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). The remaining reagents and compounds were all of analytical grade.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e4.1. Methods:\u003c/h2\u003e\n \u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e4.1.2. Experimental design\u003c/h2\u003e\n \u003cp\u003eSix groups, each consisting of ten male Wistar albino rats, were distributed at random. For seven consecutive days, rats in the normal control group received vehicle saline and CMC (both intraperitoneal and orally).\u003c/p\u003e\n \u003cp\u003eOn the 7th day, the second group of rats received 0.9% saline (i.p) for 7 consecutive days with a one single dose of paraquat (30 mg/kg, intraperitoneal) (Ahmed et al., \u003cspan\u003e2019\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003eThe 3rd group received cinnamic (50 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days with a one single dose of paraquat (30 mg/kg, intraperitoneal) on the 7th day.\u003c/p\u003e\n \u003cp\u003eThe 4th group received Silibinin (200 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days with a one single dose of paraquat (30 mg/kg, intraperitoneal) on the 7th day.(Lu et al., \u003cspan\u003e2009\u003c/span\u003e, Song et al., \u003cspan\u003e2017\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003eThe 5th group received cinnamic acid (50 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days.(Abd El-Raouf et al., \u003cspan\u003e2015\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003eThe 6th group received Silibinin (200 mg/kg, oral) dissolved in 0.5% CMC for 7 consecutive days.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e4.1.3. Collection of samples\u003c/h2\u003e\n \u003cp\u003eAll rats were sacrificed by being decapitated 48 hours after receiving a paraquat injection. Immediately after isolation, the lungs were weighed. As soon as they were ready to be processed, all samples were kept at -80\u0026deg;C.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e4.1.4. Histopathological examination\u003c/h2\u003e\n \u003cp\u003eImmediately after each animal, the upper left lung lobe was fixated in 10% neutral buffered formalin. The lungs underwent a process of progressive dehydration, paraffin embedding, sectioning into 4\u0026micro;m, and subsequent staining with hematoxylin and eosin (H\u0026amp;E). An assessment was conducted on lung specimens to identify histological changes that are indicative of ALI. These changes included interstitial edema, neutrophil infiltration, alveolar congestion, and hemorrhage. A pathologist conducting the histopathological analysis was not informed of the experimental design employed in this investigation.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e4.1.5. Preparation of lung tissue homogenates\u003c/h2\u003e\n \u003cp\u003eUsing a variable-speed homogenizer (tissue homogenizer yellow line D118 basic, Germany), the right lung tissues of the rats were immediately extracted and promptly homogenized in biphosphate buffer (pH 7.4) at concentration 0.09% at 4\u0026deg;C. by centrifuging lung homogenates (bench top cooling centrifuge model 2-16KL, Germany) for 15 min at 1000 g, 4\u0026deg;C, supernatants were obtained to be used in assessing oxidative stress-related parameters and hydroxyproline.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e4.1.6. Determination of hydroxyproline in lung tissue homogenate by ELISA\u003c/h2\u003e\n \u003cp\u003eThrough the measurement of hydroxyproline levels, the collagen content of lung tissue can be determined. The presence of collagen deposition in the lungs is suggestive of lung fibrosis. The CUSABIO kit catalog number used for carrying out the assay was CSB-E08838r. The competitive inhibition of enzyme immunoassay method is used in this assay. An antibody that is specific to Hyp has been pre-coated onto the microtiter plate that is included in this kit. Standards or samples are put into the corresponding wells of the microtiter plate using Hyp-conjugated Horseradish Peroxidase (HRP). The competitive inhibition reaction is initiated when Hyp conjugated with HRP is combined with Hyp in the samples. The color that results from adding a substrate solution to the wells is inversely proportional to the quantity of Hyp present in the sample. The process of color development stops, and the color\u0026apos;s intensity is assessed.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e4.1.7. Determination of oxidative stress biomarkers in lung homogenate:\u003c/h2\u003e\n \u003cp\u003eThe activity of MDA and TAC is determined by inhibiting chromagen reduction; this was accomplished using colorimetric assays obtained from Biodiagnostic (cat. no. TA 25 13., CAT. no. MD 25 29). The MDA assay reagent operates by utilizing the reaction between MDA and thiobarbituric acid in the sample to produce an MDA-TBA adduct. This adduct is subsequently quantified colorimetrically at an absorbance of 532 nm. The TAC assay, conversely, relies on the conversion of Cu2\u0026thinsp;+\u0026thinsp;to Cu\u0026thinsp;+\u0026thinsp;via the action of small molecule antioxidants, such as GSH. A colorimetric probe was utilized to chelate the reduced Cu\u0026thinsp;+\u0026thinsp;ion, resulting in an absorbance peak at 570 nm OD that is directly proportional to the overall antioxidant capacity.\u003c/p\u003e\n \u003cp\u003eUsing reagent CAT. No. GP 2524, reduced glutathione (GSH) levels in lung tissue homogenates were determined. The assay measures the activity of c-GPx in an indirect fashion. The reduction of an organic peroxide by c-GPx generates oxidized glutathione (GSSG), which is subsequently recycled back to its reduced state via the enzyme glutathione reductase (GR).\u003c/p\u003e\n \u003cp\u003eThe colorimetric assay for catalase is performed using biodiagnostic kit CAT. No. CA 25\u0026thinsp;\u0026minus;\u0026thinsp;17.Its fundamental principle is that catalase must react with a predetermined amount of H2O2. The reaction stops precisely one minute later when a catalase inhibitor is applied. When peroxidase (HRP) is present, the remainder of H2O2 undergoes a reaction with 3,5-dichloro-2-hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone (AAP). This reaction produces a chromophore whose wavelength of 510 nm is inversely proportional to the quantity of catalase present in the initial sample.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e4.1.8. Determination of TGF-\u0026beta;1 using ELISA\u003c/h2\u003e\n \u003cp\u003eThis assay was performed using the ER1378 FineTest biotechnology reagent (Wuhan, China). The sandwich enzyme-linked immune-sorbent assay technology formed the core of this reagent. The capture antibody was coated to 96-well plates via pre-coating. Additionally, detection antibodies were biotin-conjugated antibodies. Following the addition of the standards, test samples, and biotin-conjugated detection antibody to the wells, and to remove the mixture a wash buffer was used. After adding HRP-Streptavidin, any unbound conjugates were removed by rinsing with wash buffer. By using TMB substrates, the HRP enzymatic reaction could be observed. TMB was removed from its blue form by HRP catalysis, which was reversed to yellow upon addition of an acidic solution to stop reaction. The density of yellow color is proportional to the quantity of the target sample that is captured on the plate. Using a microplate reader to measure the O.D. absorbance at 450nm enables one to calculate the concentration of the target.\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e\u003cstrong\u003e4.Determination of phosphatidyl inositol-3-kinase (PI3K) and protein kinase B(Akt) protein expression in the lung tissue of rats by western blot analysis\u003c/strong\u003e\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eLung tissue homogenate was centrifuged in accordance with a procedure already described. The supernatant was subsequently collected, and the concentration of total protein was ascertained using the Micro BCA protein assay reagent (Cat. No: ARG66216; Arigo Biolaboratories; Rabbit pAb).\u003c/p\u003e\n \u003cp\u003eFollowing electrophoresis on 8% polyacrylamide gel, protein samples were transferred to nitrocellulose membranes.Non-specific binding sites were blocked by incubating in TBST (0.05% Tween 20 in Tris buffered saline) and 7.5% (w/v) non-fat powdered milk for two hours at room temperature. After that, membranes were cleansed with TBST for ten minutes. Subsequently, the primary specific antibodies targeting PI3K and Akt were diluted at a 1:1000 ratio and incubated at 4\u0026deg;C overnight (Cell Signaling Technology, USA). Following membrane washing, the secondary antibody is (horseradish peroxidase-conjugated anti-rabbit IgG antibody) ,it was applied at a 1:25000 dilution (Bio-Rad, USA) and incubated at room temperature for one hour. This was subsequently followed by further rinsing. Using an enhanced chemiluminescence ECL Plus System (Amersham Biosciences, USA), immunocomplexes were observed. Applying densitometry and Molecular Analyst Software (Bio-Rad, USA), the quantities were determined. The expression of PI3K and proteins was quantified in relation to \u0026beta;-actin.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e4.1.10. Determination of miRNA 193-a using qRT-PCR\u003c/h2\u003e\n \u003cp\u003eTo quantify the expression of miR193a, microRNAs were isolated using the All-in-One\u0026trade; miRNA qRT-PCR Detection Kit 2.0 (Gene Copeia TM, Rockville, USA) Cat. No. QP115 (20 RT and 200 qPCR reactions) and Cat. No. QP116 (60 RT and 600 qPCR reactions). To analyze miRNA, cDNAs containing transcribed miRNAs were amplified via PCR using the All-in-One miRNA qPCR Primer (miR-193a). Following purification with TRIzol (Invitrogen), miRNAs were transcribed into cDNAs. The primers for PCR, which are detailed in Table\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e, were subsequently utilized. For qRT-PCR, a Fast Real-Time PCR System from Applied Biosystems 7900 (Foster City, CA, USA) was utilized. As the endogenous control gene, U6 (Universal Adaptor PCR Primer) was used to normalize the input quantities. To normalize expression levels, endogenous controls were used. The determination of fold variations in expression levels was performed utilizing the 2\u0026thinsp;\u0026minus;\u0026thinsp;\u0026Delta;\u0026Delta;Ct method. The gene sequences of the PCR primers is shown in Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eeffect of cinnamic acid and Silibinin on paraquat induced lung toxicity in rats by measuring miRNA-193a-3p using qRT-PCR.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimers\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiR-193a-3p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-GTT TGG TAG CTT ATC AGA CTG A-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-GTG CAG GGT CCG AGG T-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU6 (Internal control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-CTC GCT TCG GCA GCA CA-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-AAC GCT TCA CGA ATT TGC GT-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe Effect of paraquat, Silibinin and cinnamic acid and their combination on oxidative stress markers in the lung tissue of rats.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eparameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal Control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePQ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCA\u0026thinsp;+\u0026thinsp;PQ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSilibinin\u0026thinsp;+\u0026thinsp;PQ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSilibinin\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCAT (U/gT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.9033\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0414\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8067\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.835\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.812\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAC (mM/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.9017\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.1944\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.415\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.843\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.846\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMDA (nmol/gT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9033\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8743\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5533\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3483\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.963\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGPx (U/gT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.215\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7929\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3767\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.645\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.108\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.126\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHYP (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.143\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.532\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.265\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.161\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.162\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003cem\u003eParaquat (30 mg/kg, i.p.,one single dose), Cinnamic acid (50 mg/kg, oral ), Silibinin ( 200 mg/kg oral). Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, (n\u0026thinsp;=\u0026thinsp;10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. (a) differs significantly from the normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and (b) differs significantly from the paraquat group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Statistical analysis","content":"\u003cp\u003eData were analyzed statistically by SPSS (statistical package for social sciences), version 28, IBM software (NY, USA). The data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation). SPSS was utilized to conduct one-way ANOVAs followed by Tukey post hoc tests for statistical comparisons between groups; p-values less than or equal to 0.001 considered to be significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e6.1. Histopathological examination result:\u003c/h2\u003e\n \u003cp\u003eAs shown in (Fig. \u003cspan\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eA: Normal lung tissue, the alveolar spaces were patent and lined by normal pneumocytes (black arrows) (H\u0026amp;E X 200)\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eB: Diseased lung tissue, the alveolar septa were markedly edematous, congested, infiltrated by chronic inflammatory cells (black arrow), peribronchial inflammations and epithelial desquamation (red arrow) (H\u0026amp;E X 200)\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eC: The alveolar spaces were patent and the alveolar sepata retain to the normal state with minimal residual inflammations (black arrows) (H\u0026amp;E X 100) (excellent response to toxicity).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eD: The alveolar septa were mildly edematous, congested and infiltrated by chronic inflammatory cells (black star) (H\u0026amp;E X 100) (moderate response to toxicity)\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003e6.2 Effect of paraquat, Silibinin, cinnamic acid and their combination on oxidative stress markers in lung tissue homogenates in rats:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAs displayed in (Table \u003cspan\u003e2\u003c/span\u003e) and( Fig. \u003cspan\u003e2\u003c/span\u003eA), CAT (catalase) level in the PQ group is significantly increased in comparison to the normal control group by 64.13% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), on the other hand the CA and Sil administration to PQ-treated rats significantly reduced CAT level by 73.4% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 106.6% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively in comparison to PQ group.\u003c/p\u003e\n \u003cp\u003eHYP the hydroxyproline level significantly increased in PQ group in comparison to normal control group by 271.7% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). on the other hand, the CA and Sil administration to paraquat treated rats significantly decreased HYP level in comparison to paraquat group by 26.77% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 50.19% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively, (Fig. \u003cspan\u003e2\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eGPx (glutathione peroxidase) level in the PQ group is significantly decreased as compared to the normal control group by 64.21% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), on the other hand the CA and Sil administration to paraquat treated rats significantly increased GPx level as compared to the PQ group by 73.7% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 107.5% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively, (Fig. \u003cspan\u003e2\u003c/span\u003eC)\u003c/p\u003e\n \u003cp\u003eMDA (malondialdehyde) level in the PQ group is significantly increased as compared to the normal control group by 107.3% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), on the other hand the CA and Sil administration to paraquat treated rats significantly decreased the MDA level as compared to the PQ group by 17.11% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 28.1% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively, (Fig. \u003cspan\u003e2\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eTAC (total antioxidant capacity) level in the PQ group decreases significantly in comparison to the normal control group by 63.74% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), on the other hand the CA and Sil administration to paraquat treated rats significantly increased the TAC level in comparison to the PQ group by 73.1% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 105.3% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively, (Fig. \u003cspan\u003e2\u003c/span\u003eE)\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e\u003cstrong\u003e6.3 Effect of paraquat, Silibinin, cinnamic acid and their combination on TGF-\u0026beta;1 in lung tissue homogenates in rats using ELISA\u003c/strong\u003e:\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eAs we observe in (Fig. \u003cspan\u003e3\u003c/span\u003e), paraquat significantly increased TGF-\u0026beta;1 level in the lung tissue homogenates of rats by 113.6% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 in comparison to normal control group. On the other hand, CA and Sil administration to PQ- treated rats decrease significantly the TGF-\u0026beta;1 level by 15.8% P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and 26.3% P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e\u003cstrong\u003e6.3 Effect of paraquat, Silibinin, cinnamic acid and their combination on AKT and PI3K in lung tissue homogenates in rats using western blot analysis\u003c/strong\u003e:\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eAs illustrated in (Fig. \u003cspan\u003e4\u003c/span\u003e and Fig. \u003cspan\u003e5\u003c/span\u003e), The expression of the PI3K and AKT proteins in the lung tissue of rats administered Paraquat increased significantly by 158.7% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and 193% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively, in comparison to the normal control group. On the other hand, the administration of cinnamic acid and Silibinin to PQ- treated rats showed a significant decrease in PI3K protein expression in the lung tissues of rats by 22.4% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and 22% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 respectively, and significant decrease in AKT protein expression in the lung tissues of rats by 26% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and 27.6% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 respectively.\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e\u003cstrong\u003e6.4 Effect of paraquat, Silibinin, cinnamic acid and their combination on expression of miRNA-193a-3p using qRT-PCR\u003c/strong\u003e :\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eAs illustrated in (Figure 4 and Figure 5), The expression of the PI3K and AKT proteins in the lung tissue of rats administered Paraquat increased significantly by 158.7% p \u0026lt; 0.001 and 193% p \u0026lt; 0.001, respectively, in comparison to the normal control group. On the other hand, the \u0026nbsp;administration of cinnamic acid and Silibinin to PQ- treated rats showed a significant decrease in PI3K protein expression in the lung tissues of rats by 22.4% p \u0026lt; 0.001 and 22% p \u0026lt; 0.001 respectively, and significant decrease in AKT protein expression in the lung tissues of rats by 26% p \u0026lt; 0.001 and 27.6% p \u0026lt; 0.001 respectively.\u003c/p\u003e\n \u003cp\u003e6.5 Effect of paraquat, Silibinin , cinnamic acid and their combination on expression of miRNA-193a-3p using qRT-PCR :\u003c/p\u003e\n \u003cp\u003eHere in Fig. \u003cspan\u003e6\u003c/span\u003e, We explored the effects of Silibinin and cinnamic acid on miRNA 193-a expression levels in fibrotic tissues of lung obtained from rats received one single dose of PQ (30 mg/kg). MiRNA-193a expression levels in lung tissues were assessed with qRT-PCR. MiR-193a expression levels were decreased in the lungs during paraquat-induced lung fibrosis in comparison to the normal control group by 62% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, miR-193a expression was significantly increased in cinnamic acid and Silibinin -treated rats in comparison to the Paraquat group by 64.7% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 78.5% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e6.6. Correlation between miRNA 193a and other parameters:\u003c/h2\u003e\n \u003cp\u003eIn Fig. \u003cspan\u003e7\u003c/span\u003eA ,there is a correlation between miRNA 193-a and transforming growth factor \u0026beta;1, where R= -0.96 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. This proves a very strong relationship mechanism between both parameters.\u003c/p\u003e\n \u003cp\u003ethe correlation between miRNA 193-a and serine/threonine kinase 1 (Akt) is obvious, where R= -0.94 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. This proves a very powerful relationship mechanism between both parameters, Fig. \u003cspan\u003e7\u003c/span\u003eB.\u003c/p\u003e\n \u003cp\u003eAnd at last, the correlation between miRNA 193-a and hydroxyproline, where R= -0.94 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. This proves a very strong correlation mechanism between both parameters, Fig. \u003cspan\u003e7\u003c/span\u003eC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e6.7. A graph to summarize the Mechanism of our study.\u003c/h2\u003e\n \u003cp\u003eIn Fig. \u003cspan\u003e8\u003c/span\u003e,this graph presents and illustrates our study in brief, where it shows the effect of each of paraquat, Silibinin and cinnamic acid on lung tissue of rats and pulmonary fibrosis. Also, it shows the mechanism of PI3K/AKT pathway and its relationship with miRNA 193-a, growth factor \u0026beta;1 and collagen deposition in lungs.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith over a hundred uses on crops in over 120 countries, paraquat is the third most widely used pesticide in the world. Severe lung damage and Parkinson's disease (PD) are the consequences of PQ poisoning. Self-induced poisoning is a significant public health problem in developing countries and is associated with PQ (Tsai, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Since its introduction in agriculture, accidental or deliberate ingestion has resulted in thousands of deaths each year. PQ has been identified as a significant health hazard due to its potential to cause severe lung damage in both humans and experimental animals. It has been extensively studied as a pulmonary toxicant. (Tyagi and Singh, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe prophylactic effects of cinnamic acid and Silibinin against paraquat-induced lung toxicity may be attributed to their antioxidant and anti-inflammatory properties. These compounds have been reported to scavenge free radicals, reduce oxidative stress, and inhibit the production of pro-inflammatory cytokines. Additionally, cinnamic acid and Silibinin may modulate various signalling pathways involved in oxidative stress and inflammation, such as AKT/mTOR pathway. inhibition of the PI3K/AKT pathway can enhance the expression of antioxidant enzymes and promote cellular defense against oxidative damage. Furthermore, cinnamic acid and Silibinin have been shown to possess anti-fibrotic properties, which may contribute to their protective effects against lung toxicity. These compounds can inhibit the activation of fibroblasts and the deposition of extracellular matrix components, thereby preventing tissue fibrosis.\u003c/p\u003e \u003cp\u003eIn our study, the administration of paraquat to rats resulted in the development of pulmonary toxicity, as indicated by increased levels of oxidative stress markers (hydroxyproline and lipid peroxides) in tissue homogenates.\u003c/p\u003e \u003cp\u003eThe results indicate that paraquat administration significantly decreased the levels of catalase (CAT), total antioxidant capacity (TAC), and glutathione peroxidase (GPx) in the lungs of rats. On the contrary, the paraquat group exhibited a significant elevation in malondialdehyde (MDA) and hydroxyproline concentrations when compared to the normal control group. Prior research has established that reactive oxygen species, namely hydrogen peroxide and superoxide radicals, are produced when the enzyme NADPH-Cyt-p-450 reductase metabolizes paraquat.(Yumino et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) The accumulation of superoxide radicals has the potential to induce lipid peroxidation, reduce the overall antioxidant capacity, and promote cytokine formation in lung tissues (Tyagi and Singh, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the elevated lung hydroxyproline levels in the current study may serve as an early indicator of fibrosis, as hydroxyproline is a major component of collagen (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCinnamic acid is a main constituent of cinnamon. This compound has been reported to be effective against cisplatin toxicity in rats and also to reduce the inflammatory markers as well (El-Sayed et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Silybin as well, or Silibinin, is a flavonoid that is derived from Silybum marianum, also known as Milk thistle. It has been reported that Silibinin reduces peroxidation of lipids, an indicator of oxidative lipids, in hepatocyte microsomes and isolated hepatocytes, suggesting that it may provide protection against oxidative stress (Lu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the current study pre-treatment with Cinnamic acid and Silibinin resulted in a significant increase in CAT, TAC, and GPx levels, while MDA and hydroxyproline levels decreased significantly in comparison to the paraquat group.\u003c/p\u003e \u003cp\u003ePresently, among all cytokines studied, the growth factor β family of proteins has shown the most significant effect on the deposition of extracellular matrix. TGF-β1, which is secreted in vitro as a latent precursor, stimulates the expression of fibroblast procollagen genes and the synthesis of proteins containing this protein (Xiangdong et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). TGF- β1 increased alveolar permeability of the epithelial cells in vitro through a mechanism involving intracellular glutathione depletion. Anti-TGF- β1 antibodies prevented lung injury in hemorrhaging rodents, according to another study (Shenkar et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Previous studies proved that cinnamon extracts has potent antioxidant activity and also reduced the levels of TGF-β1 activity(Barceloux, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, a previous study proved that Silibinin promoted valsartan's anti-fibrotic effect by inhibiting the TGF-β1 signalling pathway(Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This is confirmed by our results, where PQ induced TGF-β-1 activity, but pre-treatment with cinnamic acid and silibinin reduced its levels in PQ-intoxicated lungs.\u003c/p\u003e \u003cp\u003eAs confirmed from previous studies, the proteins phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) are recognized for their involvement in the processes of wound healing. Stimulation of the PI3K/AKT signaling pathway is recognized as the fundamental factor in keloid pathogenesis by inducing collagen synthesis (Kim et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Growth factors and cytokines stimulate tuberous sclerosis 1 proteins one and two in response to physiological stimuli by phosphorylating Akt and PI3K, which subsequently stimulates mTOR. And as previously described, paraquat contributes to the pathogenesis of pulmonary fibrosis by activating the PI3K/Akt pathway in alveolar epithelial cells and inhibiting autophagy activity (Jiang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Shi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), whereas silibinin has previously been shown to inactivate the PI3K/Akt pathway in kidney cancer cells (Yassin et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cinnamic acid as well inhibited the propagation of gastric cancer cells by downregulating the PI3K/Akt pathway (Li and Hu, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study we confirmed this evidence where AKT and PI3K were inhibited by Silibinin and cinnamic acid in PQ-treated rats.\u003c/p\u003e \u003cp\u003eA specific methylation pattern of miR-193a was found in patients with non-small cell lung cancer. By analysing the expression patterns of miR-193a-3p in NSCLC tissues, it was demonstrated that miR-193a-3p may act as a tumour suppressor in NSCLC and regulate the signalling of the PI3K/Akt pathway by targeting KRAS (Fan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Khordadmehr et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Another study found that miR-193a-3p inhibited lung cancer cell proliferation and invasion while promoting apoptosis (Liang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Paraquat was proved to have impact on the miRNA 193a expression by its inhibition and thus inducing oxidative stress in lung cells (Liu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). And this is similar to what we have shown in our study, the rats treated only with PQ have lower levels of miRNA 193-a expression than those treated with cinnamic acid and silibinin.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study illustrated that paraquat could induce pulmonary toxicity and fibrosis by enhancement of oxidative stress and collagen deposition in lung tissues and consequently increasing level of TGF-β1, also affecting pathway of (PI3K/Akt) by inducing it and inhibiting the signaling of (miRNA 193-a) in lung tissues. Cinnamic acid and Silibinin on the other hand offered a new prophylactic effect against PQ toxicity by downregulation of (PI3K/Akt) pathway and increasing the expression level of miRNA 193-a in the lung tissues.\u003c/p\u003e \u003cp\u003eMore studies are recommended for the synergistic effect of combination cinnamic acid and Silibinin as protective agents against lung toxicity. Investigation of the pulmonary protective effects of cinnamic acid and Silibinin in patients intoxicated with paraquat is recommended through clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgement:\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their appreciation for EL NAHDA university animal house, Dr. NM Ahmed \u0026nbsp; and Dr. Mira Magdy for their support in the statistical advisory and their help in the preparation of the manuscript.\u003c/p\u003e\n\u003col class=\"decimal_type\"\u003e\n \u003cli\u003eEthical approval\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eFaculty of Pharmacy of Benisuef University, Egypt, approved the current research in accordance with ARRIVE guidelines (Animal Research: Reporting of In-Vivo Experiments).This research was approved by BSU-IACUC reviewers and the approval number is (022-529).The National Institutes of Health\u0026apos;s (NIH) guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and the local institutional Research Ethics Committee\u0026apos;s approval were both followed in the carrying out of each experiment in respect to the ARRIVE guidelines (Animal Research: Reporting of In-Vivo Experiments).\u003c/p\u003e\n\u003cp\u003eConsent to participate:\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication:\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConflicts of interests:\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e\n\u003cp\u003eData availability:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbd El‐Raouf OM, El‐Sayed ESM, Manie MF (2015) Cinnamic acid and cinnamaldehyde ameliorate cisplatin‐induced splenotoxicity in rats. 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Pediatric emergency care 31: 117-121\u003c/li\u003e\n\u003cli\u003eYan B, Chen F, Xu L, Xing J, Wang X (2017) HMGB1-TLR4-IL23-IL17A axis promotes paraquat-induced acute lung injury by mediating neutrophil infiltration in mice. Scientific reports 7: 597\u003c/li\u003e\n\u003cli\u003eYassin NYS, AbouZid SF, El-Kalaawy AM, Ali TM, Elesawy BH, Ahmed OM (2021) Tackling of Renal Carcinogenesis in Wistar Rats by Silybum marianum Total Extract, Silymarin, and Silibinin via Modulation of Oxidative Stress, Apoptosis, Nrf2, PPARgamma, NF-kappaB, and PI3K/Akt Signaling Pathways. Oxid Med Cell Longev 2021: 7665169\u003c/li\u003e\n\u003cli\u003eYumino K, Kawakami I, Tamura M, Hayashi T, Nakamura M (2002) Paraquat-and diquat-induced oxygen radical generation and lipid peroxidation in rat brain microsomes. The journal of biochemistry 131: 565-570\u003c/li\u003e\n\u003cli\u003eZeinvand-Lorestani H, Nili-Ahmadabadi A, Balak F, Hasanzadeh G, Sabzevari O (2018) Protective role of thymoquinone against paraquat-induced hepatotoxicity in mice. Pesticide biochemistry and physiology 148: 16-21\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Paraquat, Silibinin, cinnamic acid, miRNA 193a, PI3K, AKT","lastPublishedDoi":"10.21203/rs.3.rs-4086459/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4086459/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eLevels of reactive oxygen species (ROS) are the primary determinants of pulmonary fibrosis. It was discovered that antioxidants can ameliorate pulmonary fibrosis caused by prolonged paraquat (PQ) exposure. However, research into the precise mechanisms by which antioxidants influence the signaling pathways implicated in pulmonary fibrosis induced by paraquat is still insufficient. This research utilized a rat model of pulmonary fibrosis induced by PQ to examine the impacts of Silibinin (Sil) and cinnamic acid (CA) on pulmonary fibrosis, with a specific focus on pro-fibrotic signalling pathways and ROS-related autophagy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eLung injury induced by paraquat was demonstrated to be associated with oxidative stress and inflammation of the lungs, downregulated (miR-193a) and upregulated PI3K/AKT/mTOR signaling lung tissues. Expression levels of miR-193a were determined with quantitative real-time PCR, protein level of protein kinase B (Akt), phosphoinositide 3-Kinase (PI3K) were determined by western blot analysis. Hydroxyproline levels (HYP) and Transforming growth factor-β1 (TGF-β1) were measured by ELISA, malondialdehyde(MDA), total antioxidant capacity (TAO), glutathione peroxidase (GSH) and catalase and were measured in lung tissue homogenates colorimetrically using spectrophotometer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eLong-term exposure to paraquat resulted in decreased PI3K/AKT signalling, decreased cell autophagy, increased oxidative stress, increased and helped pulmonary fibrosis formation. Silibinin and cinnamic acid also decreased oxidative stress by increasing autophagy and miR-193a expression, which in turn decreased pulmonary fibrosis. These effects were associated by low TGF- β1.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eSilibinin and cinnamic acid inhibited PQ-induced PI3K/AKT by stimulating miR-193-a expression, thus attenuating PQ- induced pulmonary fibrosis.\u003c/p\u003e","manuscriptTitle":"Protective effects of Silibinin and cinnamic acid against paraquat-induced lung toxicity in rats: impact on oxidative stress, PI3K/AKT pathway and miR-193a signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 09:56:18","doi":"10.21203/rs.3.rs-4086459/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-30T19:21:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-25T14:22:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254763195297377933015972812819534629959","date":"2024-06-03T07:43:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"701b88d7-a494-4295-85c7-17db887cabdf","date":"2024-04-03T08:01:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-03T07:59:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-28T05:04:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-28T05:04:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2024-03-12T16:33:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c93a2856-5017-4d07-851b-d13ca4f896d0","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:02:22+00:00","versionOfRecord":{"articleIdentity":"rs-4086459","link":"https://doi.org/10.1007/s00210-024-03511-y","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2024-10-25 15:57:37","publishedOnDateReadable":"October 25th, 2024"},"versionCreatedAt":"2024-04-01 09:56:18","video":"","vorDoi":"10.1007/s00210-024-03511-y","vorDoiUrl":"https://doi.org/10.1007/s00210-024-03511-y","workflowStages":[]},"version":"v1","identity":"rs-4086459","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4086459","identity":"rs-4086459","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}