The Restoring Effect of Human Umbilical Cord-Derived Mesenchymal Cell-Conditioned Medium (hMSC-CM) against Carbon Tetrachloride-Induced Pulmonary Fibrosis in Male Wistar Rats

Objective Pulmonary toxicity induced by CCl4, a model of idiopathic pulmonary fibrosis (IPF), leads to tissue remodeling and inflammation. Human umbilical cord mesenchymal cell-conditioned medium (hMSC-CM) is a potent anti-inflammatory, antioxidative, and antifibrotic agent. Methods Forty male Wistar rats were assigned to the control (C), olive oil control (C.O) (hMSC-CM), control (C.Ms), fibrosis (fb), and fibrosis with hMSC-CM (f.Ms) treatment groups. The groups C, C.O, and C.Ms received PBS (200 µl), olive oil (1 ml/kg), and hMSC-CM (100 μg protein/kg), respectively. The fibrosis group was administered with only CCl4 (1 ml/kg). The last group, f.Ms was treated with CCl4 (1 ml/kg) and 100 μg protein/kg IV hMSC-CM. While the treatment with olive oil and CCl4 was performed for 2 days/week from the first week for 12 weeks, the treatment with PBS and hMSC-CM was carried out 2 days/week from week 4th to week 12th. The effect of the UC-MSC culture medium treatment on the lung was evaluated by assessing lysyl oxidase (LOX), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-β1 (TGF-β1) genes, and proteins expression by real-time RCR and western blotting, respectively. Results Lysyl oxidase (LOX), tumor necrosis factor-alpha (TNF-α), transforming growth factor-b1 (TGF-β1), malondialdehyde (MDA), and oxidative stress levels were markedly higher in the fibrosis group than in the control groups (p ≤ 0.001). Additionally, glutathione (GSH) in the fibrosis group was markedly lower than those in the control groups (p ≤ 0.001). Fibrosis in the UC-MSC treatment group had milder histopathological injuries than in the fibrosis group. Conclusion hMSC-MSC as a strong anti-inflammatory, antioxidative, and antifibrotic decreases the level of oxidative stress, proinflammatory cytokines, and MDA causing a restoring effect against CCl4-induced pulmonary fibrosis.


Introduction
Idiopathic pulmonary fbrosis (IPF), such as chronic lung disease, is characterized by the excessive accumulation of myofbroblasts and extracellular matrix (ECM) proteins in the lung, which leads to compromised lung function and increased lung remodeling [1]. Despite the repair of damaged alveolar epithelial cells by infammatory and growth factors, dysregulation of the repair process leads to lung fbrosis [2]. Based on the evidence, proinfammatory factors, growth factors, and collagen deposition generated from fbroblasts causes IPF. Accordingly, the cross-linking reaction and deposition of collagen are catalyzed by lysyl oxidase (LOX) [3]. On the other hand, the release of infammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and other ligands such as transforming growth factor-β1 (TGF-β1) have regular efects on LOX levels [4]. Tere is ample research on the role of TGF-β1 in the increase in LOX levels by multiple signaling pathways and factors [3,5].
In addition, pulmonary fbrosis can be induced by the exposure to chemotherapy, radiotherapy, drugs, and environmental toxins such as carbon tetrachloride (CCl 4 ) which have toxic efects. Te harmful efects of CCl 4 are caused by the trichloromethyl (CCl 3 • ) and trichloromethyl peroxyl (CCl 3 O 2 • ) radicals. Terefore, CCl 4 radicals are known to cause lung fbrosis by lowering glutathione (GSH) and elevating lipid peroxidation, releasing cytokines such as TNF-α and growth factors such as TGF-β1. Tus, the released mediators cause increased mRNA LOX expression, fbroblastic proliferation, and ECM deposition [5][6][7].
Despite the lack of specifc treatment methods for IPF, lung transplantation and corticosteroid administration are common treatments for IPF. Although the side efects of corticosteroid therapy, such as hyperglycemia, hypertension, and electrolyte anomalies are well known [8], novel therapeutic methods with minimal side efects are needed [9,10]. Recent reports have shown the administrations of MSC (mesenchymal stem cells) as efective agents on lung repair for animal models with IPF [11][12][13]. Accordingly, in the ideal time of MSC therapy after the establishment of the IPF models (6-90 days), TNF-α and TGF-β1 levels returned to normal levels [8,[14][15][16]. According to the literature [17], MSC specializations could be better identifed at most available MSC resources for therapeutic strategies; thus, we used human umbilical cord mesenchymal cell-conditioned medium (hMSC-CM) for the experiments. Current studies have mostly used the IPF model induced by bleomycin [18,19], and a few studies have used the IPF model induced by CCl 4 [7,20,21]. To determine whether MSCs have a medicinal efect on IPF in rats, this study established an IPF model using CCl 4 in rats and assessed the efects of intravenous UC-MSC culture medium transplantation.

Te Preparation and Intravenous
Transplantation of hMSC-CM. Te culture, isolation, and purity evaluation of UC-MSC from human infant umbilical cord samples were conducted according to preliminary studies [22,23]. Briefy, after the human umbilical cord mesenchymal cells were cultured in DMEM-low glucose for 72 h, the detached and suspended cells in the fresh medium were centrifuged at 400g and 15000g for 10 min, respectively. Te supernatant obtained was used to treat the animals with pulmonary fbrosis. Tis study was approved by the Research Ethics Committee of Hamadan University of Medical Sciences (IR.UMSHA.REC.1398.1027). To assess the side efects of three doses of 100, 200, and 400 µg protein/kg/body weight by biochemical and histopathological observations, the 100 µg protein/kg/body weight dosage of hMSC-CM was equivalent to the impressive dose used in relevant investigations [17].

Animal Study.
Forty male Wistar rats weighing 180-200 g were acquired from the animal house of Hamadan University of Medical Sciences and maintained under standard conditions (22-24°C temperature, 12/12-hour light/dark cycle, free access to food and water). Considering the 12-week period for this study, the animals were randomly divided into fve groups (n � 8) as follows: (1) Group C (control group): the animals received sterile PBS (100 μl/IV, 2 days/week, beginning from the 4 th week during the 12 weeks of the experiment).

Sampling.
Twenty-four hours after the last treatment, all rats were sacrifced. Subsequently, lung tissues were separated and divided into two parts. Te frst part of the lungs was taken for histopathologic examination, while the second part was stored at −80°C for other investigations.

Histopathological Investigation.
Lung samples were fxed in 10% bufered formalin, embedded in parafn, and sliced into 5 μm thick sections. Te sections were then stained with hematoxylin and eosin (H & E) and Masson's trichrome (M'sT). Finally, the stained sections were studied using a light microscopic view of the lung tissue from diferent groups of rats for the demonstration of collagen.

Total Antioxidant Capacity (TAC) and Total Oxidant Status (TOS) Assays.
Te total antioxidant capacity (TAC) assay was conducted using the ferric reducing antioxidant power method (FRAP) [25], while the total oxidative capacity was determined by the oxidation of ferrous iron to ferric in lung tissues and the measurement of ferric by xylose orange [26]. Te division of TOS into TAC was performed to compute the oxidative stress index (OSI).

Glutathione (GSH) and Malondialdehyde (MDA) Assays.
MDA and GSH in the lung tissues were measured according to the instructions of the assay kit producer (Kiazist, Iran).

Hydroxyproline Contents Assay.
Te method described in a previous study [22] was applied to the hydroxyproline content assay of the lung samples from each rat. Lung tissues were powdered using liquid N 2 and 30 mg of each tissue was transferred into a micro tube. Ten, 100 µl dH 2 O was added to each tube and micro tubes were vortexed. After that, 100 µL HCl 12 M was added to the tubes and the tubes were vortexed. Next, the tubes were incubated for 48 h at 120°C. Te dried samples were recovered in 200 μl assay bufer and centrifuged at 12, 000 × g for 15 min. Finally, 50 ml of oxidant was added to the supernatant and incubated for 15 min at room temperature. After reacting supernatants with Ehrlich's reagent, according to the instructions of the assay kit producer (Kiazist, Iran), colored products as hydroxyproline content indexes were measured via spectrophotometry at 540 nm.

Enzyme-Linked Immunosorbent Assay (ELISA).
After the lung homogenate was prepared, the protein levels of TNF-α (pg/mg protein) were assayed using a rat TNF-α ELISA MAX ™ Deluxe set kit (BioLegend, San Diego, USA) and subsequently normalized to the total protein content.

Western Blot Assay.
Te homogenate of the lung tissue was placed in radio-immunoprecipitation assay (RIPA) lysis bufer containing a mixed protease inhibitor to obtain total proteins. Protein concentrations in the homogenates were assayed using the bicinchoninic acid (BCA) method. Specifc amounts of the protein were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, United States), and blocked with 5% BSA for 2 h before incubation with primary antibodies against TGF-β1 (ab215715, Abcam), LOX (ab174316, Abcam), and β-actin (ab119716, Abcam) as loading controls for 14 h at 4°C. After washing with Tris-bufered saline (TBST), the membranes were incubated with HRP-labeled goat anti-rabbit IgG for 1 h at room temperature, and the bound antibody was detected using an enhanced chemiluminescence detection reagent (Bio-Rad, Hercules, CA, United States). Te protein expression was quantifed by the densitometric analysis by ImageJ software [27].

Histopathological Findings.
As shown in Figure 1, rats that received normal saline in group C had a normal architecture of the lung tissue, typical alveoli, and thin alveolar walls. Furthermore, the administration of olive oil and MSCs in groups C.O and C.Ms did not afect the normal histology of the organ (the alveoli had thin walls with no evidence of fbrosis). In contrast, the lungs of rats administered with CCl 4 in group Fb showed extensive fbrosis with widespread deposition of collagen fbers throughout the organ. Te interstitial tissue proliferation led to the alteration of organ architecture, so few alveoli, if any, could be detected in the tissue. Nevertheless, rats that received CCl 4 together with MSC in the Fb.Ms group showed reduced fbrotic tissue mass, expansion of alveolar spaces, and narrowing of alveolar walls.

Biochemical Investigation.
In group Fb, the mean levels of TOS, OSI, and MDA increased (Figures 2(a)-2(c), p < 0.001) whereas TAC and GSH levels decreased signifcantly (Figures 2(d) and 2(e), p < 0.001) in comparison with groups C, C.O, and C.Ms. However, the TOS (Figure 2(a), p < 0.01), OSI, and MDA (Figures 2(b) and 2(c), p < 0.001) levels were lower in the Fb.Ms group than in the Fb group. Also, TAC and GSH levels increased signifcantly in the Fb.Ms group compared to those in the Fb group (Figures 2(d) and 2(e), p < 0.001).
After comparing the group Fb.Ms with group C, a signifcant increase was observed in the TOS (Figure 2(a), p < 0.001), OSI (Figure 2 (Figures 2(a), 2(c) and 2(e), p < 0.05).   Figure 3, pulmonary hydroxyproline content was remarkably increased in rats of group Fb compared with groups C, C.O, and C.Ms (p < 0.001). Furthermore, the pulmonary hydroxyproline content was signifcantly lower in Fb.Ms than in the group Fb (p < 0.001) but was augmented compared with groups C, C.O, and C.Ms (p < 0.001).

Discussion
In our study, the OSI and MDA levels, gene expression, and protein levels of LOX, TGF-β1, and TNF-α in the lung tissue of the CCl 4 administered group were clearly higher than those in the control groups. Histopathological investigations of the tissues in group Fb showed serious histopathological injuries, such as the widespread deposition of collagen fbers, lack of alveoli, and excessive lung tissue injury. In contrast, OSI and MDA levels, gene expression, and protein levels of LOX, TGF-β1, and TNF-α in the Fb.Ms group were noticeably lower than those in the Fb group. In the abovementioned group, histopathological damage in the lung tissues improved, the fbrotic tissue mass shrank, and alveolar spaces expanded.
In IPF models induced by toxins, such as bleomycin, irradiation, asbestos fbers, and CCl 4 , CCl 4 -induced toxicity has been proposed to result in pulmonary injury. Although the primary target organ of CCl 4 toxicity is the liver, IP injection of CCl 4 leads to the development in alveolar damage [28,29]. As olive oil is not harmful, IP and CCl 4 were dissolved in olive oil for IP application [30,31]. After inducing the model of pulmonary fbrosis with CCl 4 , it was deposited in many tissues, including the lungs. As noted, CCl 4 is metabolized by cytochrome-P450 enzyme into two metabolites, CCl 3 • and CCl 3 O 2 • [32]. Te rapid reaction of the mentioned metabolites with O 2 leads to the release of reactive-free radicals. Free radicals, via the oxidation of polyunsaturated fatty acids in cell membranes, help to develop lipid peroxidation. Te elevation of MDA levels, as a lipid peroxidation index, leads to intense pulmonary damage [33]. Furthermore, free radicals produced by CCl 4 , by inhibiting antioxidant enzymes such as glutathione peroxidase (GPX), increased oxidized GSH levels and hydrogen peroxide, and formation of reactive oxygen species (ROS). Lower GSH levels protect cell membranes against lipid peroxidation. Tus, following higher oxidative stress, overuse occurs due to reduced GSH levels. In another study, a lower CCl 4 toxicity by GSH was reported [21]. hMSC-CM prevents the impairment of cell membranes and lipid by reducing the levels of GSH and oxidized glutathione, attenuating myeloperoxidase, and increasing glutathione levels [34][35][36]. In the present study, while the MDA level in group Fb was more remarkably elevated than that in the control groups (groups C, C.O, and C.Ms), the GSH level was signifcantly lower than that in the control groups. Te GSH and MDA levels were lower in the Fb.Ms group. Terefore, low glutathione and high MDA levels of group Fb demonstrated that CCl 4 leads to ROS formation and lipid peroxidation, causing lung tissue damage. Te elevated GSH level in Fb.Ms compared to that in Fb group and the similar MDA level in the control groups have shown that MSC can prevent lung tissue damage by diminishing ROS formation and lipid peroxidation.
CCl 4 administration leads to the release of lytic enzymes and cytokines, such as TNF-α, following stimulation of the alveolar macrophages. Tese factors derived from alveolar macrophages exacerbate damage to the bronchial membrane and epithelial cells and impair pulmonary function [37][38][39]. High TNF-α levels, as one of the primary regulatory cytokines of the immune system, can elevate other proinfammatory cytokines and produce oxidant substances by encouraging the migration of neutrophils to damaged parts and releasing proteolytic enzymes derived from them [38,40]. Many studies have reported that MSC reducer TNFα level by decreasing ROS formation and lipid peroxidation [34,35,[41][42][43]. MSC enhances anti-infammatory responses by enhancing soluble interleukin (IL-1β) receptors and IL-10, followed by a decrease in the levels of interferon (IFN-c), TNF-α, and IL-2 [14].
In contrast, alveolar macrophages, as the main source of TGF-β1, play a multifunctional role in the IPF process by inducing LOX expression in fbroblasts, ECM synthesis, fbroblast proliferation, myofbroblast recruitment, and infammation promotion [44,45]. Tus, the high expression of LOX in the IPF process may increase collagen cross-linking [2,44,45]. Moreover, active fbroblasts in IPF are created through mechanisms such as epithelial-to-mesenchymal transition (EMT) [46], and TGF-β1 is a key factor in the EMT during IPF progression [47]. Hashimoto et al. reported that endothelial cells stimulate the production of numerous fbroblasts in a bleomycin-induced IPF model, and the fundamental mechanism of EMT in endothelial cells is involved in Ras and TGF-β1 activation [48]. Additionally, after the activation of TGF-β1, habitant fbroblasts can diferentiate into myofbroblasts and aggregate in the injured lung tissues [49].
MSC exert anti-infammatory and antifbrotic efects by secreting cytokine modulators [50]. In one study of bleomycin-induced fbrosis in mice, MSC improved survival by diminishing TNF-α, TGF-β1, and LOX levels, and fnally the infammation process in IPF [51]. In another study, MSC-derived growth factors were reported to play critical roles in the maintenance of lung permeability and restoration of alveolar epithelial cells following injury [52]. For example, Ionescu et al. revealed that insulin-like growth factor 1 (IGF-1), a growth factor derived from MSC, attenuates lung infammation [53]. Furthermore, according to Moodley et al., treatment with MSC led to a reduction in lung collagen deposition in the fbrosis of animal models  International Journal of Infammation [41]. It is possible that MSC reduces TGF-β1 and LOX levels, which in turn reduces collagen deposition and prevents the development of IPF [54]. Interestingly, some studies have reported that MSCs can secrete TGF-β1 [51,[55][56][57]. Tis phenomenon may explain why they exacerbate bleomycininduced pulmonary fbrosis when prescribed to mice during the acute fbrotic phase. Tis fnding conficts with the antifbrotic efects of MSCs [46]. However, Liu et al. revealed the human BM-MSCs isolated from healthy people secreted an extremely high level of TGF-β1 compared with MSCs derived from the umbilical cord, which secreted lower levels of TGF-β1 [58]. In line with our fndings, many studies have showed that the UC-MSC therapy leads to reduced gene expression and protein level of TGF-β1 in lung tissue following induced fbrosis [36,41,42,57,[59][60][61].

Conclusion
A toxic dose of CCl 4 may cause severe pulmonary fbrosis by increasing cytokine production, alveolar macrophage infltration, and ROS formation. hMSC-CM, a potent reducer of TGF-β1, LOX, and TNF-α, may have a prophylactic efect against CCl 4 -induced toxicity by reducing fbroblast differentiation, collagen deposition, ROS formation, and cytokine production.

Data Availability
All data used and analyzed during the current study are included in this manuscript and are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.