Antioxidant therapy against TGF‐β/SMAD pathway involved in organ fibrosis

Abstract Fibrosis refers to excessive build‐up of scar tissue and extracellular matrix components in different organs. In recent years, it has been revealed that different cytokines and chemokines, especially Transforming growth factor beta (TGF‐β) is involved in the pathogenesis of fibrosis. It has been shown that TGF‐β is upregulated in fibrotic tissues, and contributes to fibrosis by mediating pathways that are related to matrix preservation and fibroblasts differentiation. There is no doubt that antioxidants protect against different inflammatory conditions by reversing the effects of nitrogen, oxygen and sulfur‐based reactive elements. Oxidative stress has a direct impact on chronic inflammation, and as results, prolonged inflammation ultimately results in fibrosis. Different types of antioxidants, in the forms of vitamins, natural compounds or synthetic ones, have been proven to be beneficial in the protection against fibrotic conditions both in vitro and in vivo. In this study, we reviewed the role of different compounds with antioxidant activity in induction or inhibition of TGF‐β/SMAD signalling pathway, with regard to different fibrotic conditions such as gastro‐intestinal fibrosis, cardiac fibrosis, pulmonary fibrosis, skin fibrosis, renal fibrosis and also some rare cases of fibrosis, both in animal models and cell lines.


| INTRODUC TI ON
Fibrotic diseases account for 45% of mortality in the United States. 1 Fibrosis usually happens as a result of disruption in body's natural ability to repair scars and damages. 2 In fact, fibrosis is a phenomenon described by extreme accumulation of collagen and other extracellular matrix (ECM) components. 3Subsequent to an injury or scar, the affected area and immune cells, especially macrophages, secrete different kinds of chemokines and cytokines, as well as signalling proteins, such as TGF-β. 4Consequently, secretion of these proteins causes an increased rate of proliferation and also migration of fibroblasts to the affected area.After migration, fibroblasts are usually differentiated to myofibroblasts in order to repair damages more efficiently.Chronic inflammation and exposure to harmful substances cause over-healing and excessive repair of damaged tissues and ultimately fibrosis. 5This phenomenon is lethal if happens in vital organs, including lung, liver, heart and kidney. 5other mechanism which is involved in the fibrosis is epithelial-mesenchymal transition (EMT).Through this process, cells lose their epithelial characteristics, including apical-basal polarity and stable intercellular junction, and gain mesenchymal features such as cytoskeletal and morphological rearrangements and fibroblast-like gene signature. 6ansforming growth factor beta (TGF-β) is a protein ligand that activates a cascade of reactions by binding to serine threonine kinase receptors. 7TGF-β is involved in different cellular mechanisms.
For instance, it impedes proliferation and regulates ECM and collagen synthesis. 8Increased amount of ECM is a characteristic of most inflammatory diseases. 9Overexpression of TGF-β causes tissue fibrosis and scars in different organs. 10Upon binding of TGF-β, a group of transcription factors, called SMADs are phosphorylated.SMADs are the main signal transducers of the TGF-β family.After phosphorylation, they migrate to nucleus and in accordance with other factors, SMADs contribute to activation or inhibition of different genes. 11TGF-β is involved in cell growth and development.Moreover, it regulates inflammatory responses and preservation of resistance mechanisms against inflammation.Another aspect of TGF-β functions which is associated with human disorders is remodelling and repair process.The latter contributes to the angiogenesis and tissue regeneration. 12tioxidants are agents that could oppose adverse effects of oxidation from intracellular compounds. 13Based on their mechanism of action, antioxidants are divided into three groups, including primary, secondary and tertiary ones.Primary antioxidants act free radical scavengers; secondary antioxidants retard chain initiation; and tertiary ones mainly repair damaged biomolecules.A generalized function of almost all types of antioxidants is reversing the effects of ROS in cells. 14Both endogenous and exogenous antioxidants have essential roles in maintaining an optimum cellular function.Reactive oxygen species (ROS) and different nitrogen and sulfur-based compounds, which are normally generated in cells as a result of different interactions and reactions, are potentially harmful substances and could damage cells at DNA, lipid, protein, carbohydrate and other levels. 15Thus, a proper and balanced diet enriched with antioxidants could actually reverse detrimental effects of these agents.An important instance of endogenous cellular antioxidants is glutathione, which is composed of three amino acids, including glutamine, cysteine and glycine.This antioxidant confronts adverse effects of hydroperoxide and other peroxides, with the help of glutathione peroxidase (GPX). 16At molecular level, antioxidants reduce the cellular levels of free radicals through inhibition of activity or expression of free radical producing enzymes, including NAD(P)H oxidase and xanthine oxidase (XO).Besides, antioxidants can enhance the activity and expression of antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT) and GPX. 17 Vitamin C (ascorbic acid) is a great example of exogenous agents, which helps with iron absorption and reversing free radical effects. 18In recent years, antioxidants are widely used in order to prevent and treat different human disorders, especially in animal models.Some of them, including edaravone and Nacetylcysteine gained the right permission for clinical use. 19The road ahead of using these agents as therapeutic elements is long, but daily experiments are conducted on different cell lines and animal models, in order to potentiate their therapeutic properties.
In this study, we aim at reviewing the role of different kinds of antioxidants as therapeutic agents in organ fibrosis, with regard to TGF-β/SMAD pathway as the main target of antioxidants.

| OXIDATIVE S TRE SS AND ITS ROLE IN ORG AN FIB ROS IS
Oxidative stress is a condition that arises when there is an imbalance between the production of ROS and antioxidant defence mechanisms in the body. 20ROS are highly reactive molecules that are generated as a byproduct of normal cellular metabolism, but they can also be produced in response to external factors such as toxins, radiation and infections. 21,22Oxidative stress can cause damage to proteins, lipids, and DNA, leading to cellular dysfunction and tissue damage. 23S can stimulate the activation of fibroblasts, 24 which are cells responsible for the production of ECM proteins such as collagen, fibronectin and elastin. 25In addition, ROS can also stimulate the release of pro-inflammatory cytokines and growth factors that promote fibroblast activation and ECM deposition. 26Moreover, ROS can inhibit the activity of matrix metalloproteinases (MMPs), which are enzymes that degrade ECM proteins, thereby impairing the process of ECM turnover and promoting fibrosis. 27,28 the context of organ fibrosis, oxidative stress plays a critical role in the initiation and progression of the fibrotic process, 29 which is characterized by the excessive deposition of ECM proteins, and leads to the progressive loss of organ function. 30Several studies have shown that oxidative stress can promote fibrosis by inducing the activation of fibro-genic cells such as fibroblasts and myofibroblasts. 31idative stress can activate several signalling pathways that contribute to fibrosis, including transforming growth factor-beta (TGF-β). 32TGF-β is a potent inducer of ECM synthesis and is known to promote the differentiation of fibroblasts into myofibroblasts, which are the primary producers of ECM proteins. 33Moreover, oxidative stress can also contribute to fibrosis by impairing the function of antioxidant defence mechanisms. 34Studies have shown that antioxidant levels are reduced in fibrotic organs, and this reduction is associated with increased oxidative stress and fibrosis. 35erall, oxidative stress plays a critical role in the pathogenesis of organ fibrosis by promoting fibro-genic signalling pathways and impairing antioxidant defence mechanisms.Therefore, targeting oxidative stress may represent a potential therapeutic strategy for the prevention and treatment of fibrotic diseases.

| ANTI OXIDANTS A S A MODUL ATOR O F TG F-Β/ S MAD PATHWAY IN D IFFERENT ORG AN FIB ROS IS
The TGF-β/SMAD signalling pathway plays a key role in the development and progression of organ fibrosis by promoting fibroblast activation and ECM protein accumulation. 36Antioxidants have been proposed as potential modulators of the TGF-β/SMAD pathway in various organ fibrosis conditions.Here are some examples:

| Gastro-intestinal fibrosis
Liver fibrosis refers to accumulation of ECM components, especially collagen in the liver, which ultimately results in liver failure or cirrhosis.Most of the time, the treatment includes liver transplant. 37fferent kinds of fibrogenic cytokines are involved in liver fibrosis, and TGF-β is one of them. 38 this section, we inquire role of different antioxidants as regulators of TGF-β/ SMAD in gastro-intestinal fibrosis, with an especial focus on liver fibrosis (Table 1).
As shown in Table 1, most of the studied agents, had an inhibitory role on TGF-β/ SMAD pathway.As it was mentioned before, TGF-β/SMAD stimulation contributes to increased rate of ECM synthesis.Ferulic acid (FA) is a plant derived antioxidant and a free radical scavenger (a primary antioxidant) with proven beneficial roles in different conditions, especially cancer and inflammatory diseases. 66 a study conducted by Mao mu and colleges in 2018, it was demonstrated that treatment with FA could reverse the effects of carbon tetrachloride (CCl4) induced liver fibrosis. 43More specifically, treatment with TGF-β causes an overexpression of α SMA, FN, Col-I, Smad-2/3, p38 and JNK.Treatment of LX-2 cells with 30 μM (50 μM was cytotoxic) of FA reversed the effects of TGF-β in these cells 43 (Figure 1A).
Honokiol is an antioxidant with a poly-phenolic nature, mostly derived from magnolol and root bark, with protective properties against neural damage, anxiety and a variety of cognitive disorders. 67It has been shown that treatment of male SD rats with concanavalin-A, elevated alpha smooth muscle actin (α-SMA) levels in liver, which is a fibrosis indicator in most cases. 47After treatment with Honokiol for 4 weeks, reduced expression of (α-SMA) was seen. 47Additionally, it was documented that Honokiol acts as TGF-β/SMAD/MAPK inhibitor and protects against liver fibrosis 47 (Figure 1B).
In the category of flavonoids, Isorhamnetin (IsoR) belongs to class of Flavonols and is mostly found in Hippophae rhamnoides L fruits. 68oR play a role in protection against various conditions, such as cardiac and cerebral related complications. 68This agent could reverse liver fibrosis with a similar mechanism as FA. 64sed on a study performed by Tengfei Liu and colleges, it was shown that copper-based nanoparticles could act as antioxidant by scavenging ROS and reducing inflammation both in vitro and in vivo. 69Interestingly, their antioxidant property is at stake in case of protection against liver fibrosis.In a concise study, it was proved that treatment of Mononuclear Cells (MNCs) with copper-based nanoparticles, induces TGF-β/SMAD pathway and subsequent elevation of ECM compounds occurs, leading to liver fibrosis.Copperbased nanoparticles are the only exception of antioxidants that could activate TGF-β pathway and contribute to formation of fibrotic tissue.
The result of inflammatory bowel disease (IBD) in both forms, including ulcerative colitis (UC) and Crohn's disease (CD), is mostly intestinal fibrosis.Similar to liver fibrosis, excessive amount of scar tissue and over repaired damage is the cause. 70Two independent studies confirmed the role of antioxidants against intestinal fibrosis.Firstly, it was demonstrated that treatment of intestinal epithelial cells (IEC-6) with Curcumin (CUR), had two significant results: (1).inhibition of TGF-β1 induced SMAD pathway and downregulation of α-SMA and (2).Increased expression of peroxisome proliferator-activated receptor γ (PPARγ) and its nuclear localization, which inhibits EMT and protects against fibrosis 71 (Figure 1C).
Another study also confirmed the role of Calycosin (CA), which is a component of astragalus membranaceus, in fighting against intestinal fibrosis. 72This protection is done through the same mechanism as CUR, but the different is that CA does not interfere with EMT, instead, it elevates Smad7 expression, which is an inhibitory Smad and inhibits TGF-β signalling. 72Further, The polyphenol glabridin, which is derived from Glycurrhiza glabra (licorice) roots, has been utilized in traditional medicine and is known to have a variety of biological actions, including anti-inflammatory, antioxidant, and anti-fungal effects. 73One of the oral conditions that may be malignant is known as oral submucous fibrosis (OSF).By inhibiting TGF/smad signalling, glabridin prevents myofibroblast activation in human fibrotic buccal mucosal fibroblasts 73 (Figure 1D).
Table 2 shows the effects of different antioxidants in regulation of TGF-β/ SMAD pathway in other gastrointestinal disorders.

| Lung fibrosis
Pulmonary or lung fibrosis (PF) refers to progressive lung scarring with potential life-threatening properties. 79There has been an incremental trend In pulmonary fibrosis in recent years. 80Different risk factors such as smoking, exposure to harmful substances and diabetes could ultimately lead to fibrosis of the lungs. 81duction of TGF-β/SMAD pathway by antioxidants in PF was seen in multiple researches.In 2016, it was observed that treatment of A549 cells with Bleomycin (BLM), a cancer related antibiotic, 82 could induce TGF-β/ SMAD pathway, and this was concluded based on lower levels of E-cadherin and elevated levels of vimentin after treatment with BLM. 83Additionally, respiratory exposure to nanoparticulate titanium dioxide (nano-TiO2) has proven to be detrimental to lungs by elevating expression of proteins such as TGF-β1 and HIF-1α and conversely downregulating phosphorylated glycogen synthase kinase-3β (p-GSK-3β). 84 It is worth mentioning that certain vitamins, such as vitamin C and D act as antioxidants.In relation to respiratory fibrosis and inflammation, a great number of studies are about asthma and its complications.

Model of study
Research has shown that ovalbumin (OVA) induced asthma in murine models is reversed by intraperitoneal injection of vitamin D in its active form, 1, 25-dihydroxyvitamin D3. 87 It was shown that vitamin D activates Nrf2/HO-1 pathway, which itself activates gene expression of heme oxygenase-1 (HO-1), an anti-oxidative stress agent. 88Moreover, vitamin D acts as a TGF-β/SMAD inhibitor in asthmatic airway. 87 it is evident in Table 2, BLM and lipopolysaccharides (LPS) are used for induction of pulmonary fibrosis, and in most cases, treatment with different substance reverses this process by inhibiting TGF-β/ SMAD and other inflammatory pathways.On the most recent studies centered around this has been done by Yao y and colleges in 2022. 89ll this day, more than 1500 of Diterpenoid alkaloids (DAs) have been extracted from different plants.Some of these alkaloids have shown cytotoxic effects on cancer cells. 90

| Renal fibrosis
Renal fibrosis which manifests itself with tubulointerstitial fibrosis and glomerulosclerosis, is the end stage of chronic kidney disease. 113Similar to other fibrotic conditions, deposition of ECM in different areas of kidney, interrupts with its structure and function. 113It has been proved that TGF-β plays a critical role in developing renal fibrosis, either by induction of apoptosis or EMT. 114eanolic acid (OA) is natural pentacyclic triterpenoid and is found in medicinal herbs and oils, specially olive oil. 115In an in vitro study performed in QZG cells in 2010, it was shown that OA has antioxidant activity by scavenging free radicals via increasing glutathione synthesis. 116Conveniently, it is demonstrated that oral administration of OA to male SD rats for 21 days, results is reduced expression of TGF-β and its related receptors (I and II), as well as optimum levels of serum creatinine 117 (Figure 2B).
While drugs may cause certain adverse effects, they can also have positive impacts.Losartan is a good example.Sold under the brand name 'Cozaar', losartan is widely used to treat high blood pressure via inhibiting angiotensin receptor. 118Alongside with its antihypertensive activity, a clinical trial conducted in 2013 has proven that treatment of patients undergoing haemodialysis with losartan not only increases thiol groups with antioxidant activity, but also reduces Oxidative stress index (OSI). 119The exact molecular mechanism of this phenomenon was discovered by studying mice models of Renal Interstitial Fibrosis (RIF).It was demonstrated that losartan treatment acts as an anti-fibrosis agent by increasing inhibitory Smads, that is, Smad 7, and induction of TGF-β receptor (I) breakdown. 120 case of high glucose exposure to simulate glucose-mediated fibrosis, Human renal proximal tubule epithelial cell line (TH1) was treated with D-glucose for a period of time. 121Subsequent treatment with melatonin (MEL), a sleep-inducing hormone, was attentive.It was shown that MEL protects against high glucose induced renal fibrosis, by blocking TGF-β expression and subsequent phosphorylation of Smads by it. 121Additionally, MEL increased Cellular prion protein (PrPC) expression via Akt activation. 121These interventions made by MEL ultimately results in protection against fibrosis.
Similar to liver and lung fibrosis, induction of TGF-β/ SMAD pathway by antioxidants is also seen in renal fibrosis.Baicalin is a type of flavonoid compound and is derived from the root of Scutellaria baicalensis Georgi plant.This plant is a member of Lamiaceae family and is known for its medicinal properties. 122High dosage intake of Baicalin by Sprague-Dawley (SD) rats was shown to have detrimental effects on renal tissue. 123Based on dosage, Baicalin activates TGF-β/Smad signalling pathway and contributes to renal fibrosis. 123though it's not always the case, since a study conducted on mice models of RIF showed opposite results. 124It was revealed that baicalin acts as an anti-fibrotic agent in renal fibrosis via suppressing the mentioned pathway. 124ble 4 summarizes treatment of renal fibrosis models and cell lines with different antioxidants and their subsequent impact on TGF-β/ SMAD pathway.

| Cardiac fibrosis
Cardiac fibrosis is seen in different cardiomyopathies, and similar to other fibrotic conditions, is characterized by deposition of ECM in heart tissues, which ultimately results in heart failure and death. 143A suitable disease model for studying cardiac fibrosis, is induced Diabetic Cardiomyopathy (DbCM).5][146] By applying a high-fat diet and streptozotocin, induction of cardiomyopathy was achieved in all three studies.Matrine has shown anti-fibrotic properties in hepatic fibrosis, 147 and in DbCM, it protects against fibrosis and recovers left ventricular (LV) by inhibiting TGF-β1/R-Smad signalling pathway, although it does not affect levels of nhibitory Smads like Smad7. 146Dapagliflozin (DAPA) acts as sodium-glucose cotransporter 2 (SGLT2) inhibitor, and by reversing endothelial to mesenchymal transition (EndMT), it reverses fibrotic features and indexes. 145Additionally, DAPA is capable of impeding TGF-β/Smad signalling pathway via AMPKα 145 (Figure 2C).Finally, Empaglifozin (EMP), which is also a SGLT2 inhibitor, inhibits TGF-β/ SMAD pathway and on the other hand, contributes to protective effects in cardiac fibrosis by activating Nrf2/ARE signalling. 144Other components and their related mechanism in protection against cardiac fibrosis are listed in Table 5.

| Skin fibrosis
Skin occurs as a result of different skin related conditions, such as scleroderma and eosinophilic fasciitis. 152LG283 is a synthetic compound and is derived from CUR. 153 After induction of skin fibrosis by BLM in C57BL/6 mice, skin was thickened and fibrotic changes at molecular level was observed. 154Treatment with LG283 reduces capillary vessels and softens skin tissues, and also lowers α-SMA and phosphorylated Smad3 expressions. 154leroderma, also known as systemic sclerosis, is connective tissue disorder and is characterized by skin stiffness and vascular abnormalities in internal organs.This disease is categorized as autoimmune, and its exact molecular mechanism is yet to be found. 155cording to medchemexpress, DZ2002 is an immunosuppressive type III SAHH inhibitor and has beneficial properties for lupus syndrome and systemic sclerosis, only for research purposes.Research has found out that DZ2002 reduces psoriasis-like skin lesions and inflammation by regulating GATA3 methylation. 156terestingly, DZ2002 has proven to be protective in mice models of systemic sclerosis 157 (Figure 2D).The mechanism of action involves reduction of TGF-β-1 and CTGF, as well as VEGF which is responsible for vascular complications. 157ble 6 summarizes application of different compounds for treatment of skin fibrosis.

| Other fibrotic conditions
In addition to the more common cases of fibrosis mentioned earlier, there are also some uncommon instances that have not been extensively researched.

Muscle fibrosis usually occurs in dystrophies especially
Duchenne muscular dystrophy (DMD) and effects a significant portion of patients.In other cases such as muscle injuries, muscle fibrosis rarely occurs. 160V refers to formation of new blood vessels in choroid layer of the eye and could lead to vision loss.Risk of this condition increases with age, and is more prevalent in individuals over the age of 75. 161R is a complication that can occur after a person has undergone surgery for a retinal detachment.It is characterized by the growth of abnormal tissue on the surface of the retina, which can cause the retina to detach again. 162esented in some cases lumbar spinal surgery patients, Epidural Fibrosis causes a great pain.It has been shown that Scar formation and excessive ECM in epidural space is the main cause. 163l of these four conditions are studied in vivo, and it has been shown that except for muscle fibrosis, Pirfenidone (PFD) for CNV, Artesunate (ART) for PVR and Taurine (Tau) for Epidural Fibrosis could act as anti TGF-β/ SMAD and they have the potential to TA B L E 6 Skin fibrosis.

| DISCUSS ION
TGF-β/SMAD signalling pathway regulates various cellular functions, such as proliferation, differentiation and apoptosis. 167However, uncontrolled activation of this pathway leads to the accumulation of ECM compounds, resulting in organ fibrosis. 10Organ fibrosis has important health effects leading to loss of organ function, particularly in liver, lung, kidney and skin.Approximately, one third of natural deaths worldwide is attributed to organ fibrosis and subsequent loss of function of mentioned organs. 168Several studies have shown that antioxidants can efficiently reverse the effects of TGF-β/SMAD pathway in fibrosis, thus contributing to the protection against fibrosis.Although several antioxidants belonging to all three classes of antioxidants have modulating effects on the expression and activity of TGF-β/SMAD pathway, scavengers are mostly appreciated in this regard.
A generalized function of almost all types of antioxidants is reversing the effects of ROS in cells. 14By reducing ROS levels, antioxidants contribute to inhibition of fibrosis.Additionally, antioxidants can down-regulate expression of TGF-β and its receptors, and also regulate downstream molecules such as Smads, especially Smad7, and ultimately inhibit the mentioned pathway.
Several antioxidants have shown promising effects in the prevention of organ fibrosis.For instance, N-acetylcysteine (NAC) is a precursor of glutathione, which is an important antioxidant in the body.NAC has been shown to inhibit TGF-β induced profibrotic responses and inhibit lung fibrosis. 169Vitamin E as another antioxidant also has inhibitory effects on TGF-β/SMAD signalling pathway. 170 we concluded in this article, antioxidant therapy has several benefits over other treatment options for fibrosis.Unlike other treatments, which often target specific aspects of fibrosis and are invasive in nature, antioxidants have a wider impact and disrupt multiple pathways involved in fibrosis.Additionally, antioxidants are relatively safe and are considered as a non-invasive method for treatment, both in vivo and in vitro, making them a great factor for further studies.

F I G U R E 1 2
Schematic diagram illustrating the mechanism of action for antioxidants in regulating TGF-β/SMAD signalling pathway and preventing Gastro-Intestinal Fibrosis.The effects of different antioxidants in regulation of TGF-β/ SMAD pathway in other gastrointestinal disorders., FN-1, Col-I/III, Smad-2/3, IL-6, TNF-α, Ll-1β In an animal model of CP, treatment with piperine could act against fibrosis severity treatment of PF models and cell lines with different antioxidants and their impact on TGF-β/ SMAD pathway.

F I G U R E 2
Antioxidants as potential therapeutic agents: a visual overview of their role in modulating TGF-β/SMAD pathways across different fibrosis types.
to treat organ fibrosis by interrupting the TGF-β/SMAD signalling pathway in animal models and cell lines.However, additional research should be conducted in a greater extent and with more precision, to optimize the dosage and evaluate its effectiveness in various stages of fibrosis with different etiologies.Antioxidant therapy could provide a secure and efficient treatment option for fibrosis in future.AUTH O R CO NTR I B UTI O N SSoudeh Ghafouri-Fard: Validation (equal); writing -original draft (equal).Arian Askari: Validation (equal); writing -review and editing (equal).Hamed Shoorei: Investigation (equal); methodology (equal).Mohammad Seify: Formal analysis (equal); investigation (equal); TA B L E 7 Other fibrotic conditions., PAI-1, TIMP-1, Nrf-2 In mdx mice, treatment with SFN by suppressing the Nrf2mediated mentioned pathway could act against muscle fibrosis.with ART could act against the development of PVR by inhibiting the mentioned pathway and EMT process., Col-III, Smad-2/3/7, TGFβRI/RII Treatment with Tau could act against epidural fibrosis induced by laminectomy.