Chlorogenic Acid Alleviates Inflammation and Fibrosis in a Murine Model of Bleomycin-Induced Systemic Sclerosis: A Histological Analysis

Abstract

Background/Objectives: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by cutaneous and visceral fibrosis, vascular alterations, and a persistent inflammatory response. Despite advances in understanding the pathogenic mechanisms underlying SSc, current therapeutic options remain limited. Chlorogenic acid (CGA) is a polyphenol widely distributed in plants and has shown antioxidant, anti-inflammatory, and antifibrotic properties. However, its therapeutic potential in SSc has not been investigated yet. Methods: A model of SSc was established by administering bleomycin (BLM) at 100 U/kg to CD1 mice via an osmotic minipump. After fourteen days of BLM administration, CGA (60 mg/kg) was intragastric administered on consecutive days until day 20. On day 21, all mice were sacrificed. The effect of CGA was histologically evaluated by hematoxylin and eosin and Masson’s trichrome staining. Results: CGA treatment significantly attenuated dermal fibrosis in the BLM-induced mice model of SSc by reducing histopathological damage, including increased dermal thickness, inflammation, collagen deposition, and SSc-associated pulmonary fibrosis. Conclusions: The evidence shows that CGA attenuates BLM-induced SSc in a mice model and strongly suggests that CGA may be a promising compound for the treatment of SSc.

1. Introduction

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by cutaneous and visceral fibrosis, vascular alterations, and a persistent inflammatory response [1]. It has a prevalence of approximately 17.6 cases per 100,000 persons per year and an incidence of approximately 1.4 per 100,000 persons per year [2]. It occurs more frequently in female patients, with a female-to-male ratio of approximately 4:1. The disease usually manifests between the ages of 40 and 50 years but can appear at any age [3,4]. The pathophysiology of SSc is complex and multifactorial, involving an aberrant interaction between the immune system, fibroblasts, and endothelial cells. The pathogenic process begins with vascular dysfunction and endothelial damage, leading to the activation and recruitment of immune cells. These cells release cytokines and growth factors such as IL-6, transforming growth factor beta (TGF-β), and platelet-derived growth factor (PDGF), which stimulate fibroblasts to proliferate and produce excessive amounts of extracellular matrix (ECM), mainly collagen [1]. This abnormal collagen deposition leads to skin and internal organs fibrosis, affecting the function of vital structures such as the lungs, heart, kidneys, and gastrointestinal tract [5]. In addition to fibrosis, SSc is characterized by an obliterative vasculopathy affecting small arteries and capillaries, contributing to pulmonary hypertension and ischemic phenomena in the affected organs [1,6]. Chronic inflammation and autoimmunity are other critical components of the disease, with specific autoantibodies such as antitopoisomerase 1, anticentromere, and anti-RNA polymerase III antibodies used as diagnostic and prognostic markers [1,7]. Despite advances in understanding the pathogenic mechanisms underlying SSc, current therapeutic options remain limited and, in many cases, ineffective in halting the disease progression [8,9]. Therefore, the search for new therapies that can modulate these pathological processes is of vital importance.

Chlorogenic acid (CGA) is a phenolic compound widely distributed in plants and is well known for its antioxidant, anti-inflammatory, antimicrobial, anticancer, and antifibrotic activity [10,11]. Numerous studies have documented the beneficial effects of CGA in various inflammatory and fibrosing disease models [11]. For example, recent research has shown that CGA protects against hyperglycemia-induced cardiac fibrosis in a murine model of streptozotocin-induced type I diabetes [12]. In addition, it was shown that CGA was able to prevent renal fibrosis in a murine model induced by unilateral ureteral obstruction (UUO); this effect was achieved by reducing the levels of collagen fibers and that of alpha-smooth muscle actin (α-SMA), a marker of myofibroblasts [13]. It was also shown that CGA can reduce liver fibrosis associated with non-alcoholic steatohepatitis in a murine model induced by a diet deficient in methionine and choline [14]. In addition, CGA has demonstrated a remarkable ability to mitigate skin damage associated with systemic lupus erythematosus (SLE) in the MRL/lpr murine model. This therapeutic effect is attributed to the significant reduction in pathological scores related to acanthosis and cutaneous hypertrophy [15]. This available evidence suggests the therapeutic potential of CGA in pathological conditions, such as SSc.

Our study investigated the impact of CGA on inflammation and fibrosis using a bleomycin (BLM)-induced murine model of SSc. BLM is a chemotherapeutic agent that, when administered in animals, reproduces many of SSc’s clinical and pathological features, including excessive collagen production and inflammatory cell infiltration in the skin and other organs [16,17,18]. Our findings demonstrate, for the first time, the protective effects of CGA on characteristic histological skin and lung alterations in SSc, such as inflammation and fibrosis. These results may contribute to the development of new therapeutic strategies for SSc, a disease that represents a considerable challenge for both patients and healthcare professionals.

2. Materials and Methods

2.1. Animals

A total of twenty male CD1 mice, aged between 6 and 8 weeks and weighing between 34 and 42 g, were acquired from the Laboratory Animal Production and Experimentation Unit of the Center for Research and Advanced Studies of the National Polytechnic Institute (UPEAL-CINVESTAV-IPN). Rodents were subjected to a two-week acclimatization period, fed ad libitum, kept in a pathogen-free environment, and housed under controlled conditions, with 12 h light/dark cycles and at a constant temperature of 22 ± 3 °C. All experimental procedures were performed by the ethical standards for animal research, with the approval of the Institutional Animal Care and Use Ethics Committee (IACUC) of the Faculty of Medicine and Surgery of the Autonomous University Benito Juárez of Oaxaca (registration number 0047-CEI-2022).

2.2. Experimental Protocol

A mouse model of SSc induced by continuous BLM administration using mini osmotic pumps was used [17,19]. Twenty mice were randomly assigned to four experimental groups (n = 5 animals per group): The control group (CT) did not receive any stimulus, one group was only treated with CGA (CGA), another group received BLM (BLM), and the last group was subject to BLM and CGA (BLM + CGA). Mice of the BLM and BLM + CGA groups were anesthetized with isoflurane and implanted subcutaneously in the scapular region with an osmotic minipump (ALZET 1007D; DURECT, Cupertino, CA, USA) releasing BLM at a dose of 100 U/kg (BLEOCEL, Celon Labs, Hyderabad, Telangana, India) at a rate of 0.5 μL/h for seven days, designating this day as day 0. Mice of the CT and CGA groups received a sham procedure under isoflurane anesthesia. On day 10, the osmotic minipumps were removed from mice in BLM and BLM + CGA groups according to the manufacturer’s instructions. In contrast, mice of the CT and CGA groups underwent sham removal. Starting on day 14, CGA (C3873, Sigma Aldrich, St. Louis, MO, USA) was administered daily intragastrically at a dose of 60 mg/kg in sterile water to the CGA and BLM + CGA groups, continuing until day 20. On day 21, the animals were sacrificed under anesthesia, and lung and skin tissue samples were collected. Skin samples were obtained from the dorsal area, approximately 2 cm posterior to the mini osmotic pump implantation site. A schematic representation of the SSc model is shown in Figure 1.

2.3. Tissue Collection and Processing

The collected skin and lung tissue samples were immediately washed with cold phosphate-buffered saline (PBS 1X, pH 7.4), and then, they were fixed in 4% formaldehyde. Subsequently, tissue was dehydrated using a series of ethanol and xylol concentrations, including ethanol (70%, 80%, 90%, and 96%), xylol/ethanol (1:1), and xylol immersions, each for 1 h. Then, tissues were immersed in three paraffin baths at 56 °C for 1 h each to prepare paraffin blocks for histological sections. For histological analysis, tissue sections of five microns thick were obtained using a microtome (Leica, model RM 2125 RTS).

2.4. Hematoxylin and Eosin (H&E) Staining

To evaluate the overall cellular structure and tissue morphology as well as histological alterations induced by BLM, skin and lung tissue sections were stained using hematoxylin and eosin (H&E). After deparaffinization at 56 °C, slides were rehydrated by immersion in xylol, xylol/ethanol (1:1), and decreasing concentrations of ethanol (96%, 90%, 80%, and 70%) for 30 s each, followed by rinsing with water. Subsequently, the slides were stained with Harris hematoxylin (738, HYCEL, Jalisco, Mexico) for 10 min. Then, slides were immersed in an acidic ethanol solution for fixation and in an ammonia solution, followed by staining with yellow eosin (688, HYCEL, Jalisco, Mexico) for 10 min. Finally, tissue sections were dehydrated using increasing concentrations of ethanol and xylol and then mounted on synthetic resin for analysis. Images were captured using a CARL ZEISS Primo Star optical microscope with magnifications of 4× and 10× for skin and 20× for lung. Images were analyzed using ImageJ v.2.3.0/1.53t software (U.S. National Institutes of Health, Bethesda, MD, USA) to quantify dermal thickness in the skin, which was determined by measuring the average distance between the epidermal–dermal junction and the dermal junction–subcutaneous fat layer. Dermal thickness was calculated by randomly selecting five areas within each field (images captured at a magnification of 4×). A total of five randomly selected fields were analyzed for each sample. The average dermal thickness in the five areas of each field was used to obtain a final average value per sample.

Two independent investigators, blinded to the experimental design, semi-quantitatively classified inflammatory infiltrates in H&E staining of skin using an inflammation severity scale described by Gallet et al. [20]. A severity score was established from 0 to 3, where Grade 0 indicates no inflammatory infiltrate, Grade 1 some inflammatory elements, Grade 2 frequent inflammatory infiltrate, and Grade 3 ubiquitary inflammatory infiltrate.

Lung tissue stained with H&E was analyzed using ImageJ v.2.3.0/1.53t software (U.S. National Institutes of Health, Bethesda, MD, USA) to determine the percentage of alveolar space. The percentage of alveolar area was calculated by randomly selecting 20 areas within each field (images captured at 20× magnification). The mean alveolar area percentage in the 20 areas was used to obtain a final mean value per sample.

2.5. Masson’s Trichrome Staining

To assess the degree of fibrosis and collagen accumulation induced by BLM, Masson’s trichrome staining of skin and lung tissue sections was performed using the HT15 kit (Sigma-Aldrich, St. Louis, MO, USA). After deparaffinization at 56 °C, slides were rehydrated by immersion in xylol, xylol/ethanol (1:1), and decreasing concentrations of ethanol (96%, 90%, 80%, and 70%) for 30 s each, followed by washing with water. The slides were then immersed in Bouin’s solution at 56 °C for 1 h. They were then stained with Weigert’s ferric hematoxylin for 20 min, washed with water, and stained with Biebrich’s scarlet acid fuchsin for 10 min. Subsequently, slides were treated with phosphotungstic acid and phosphomolybdic acid for 20 min, rinsed, and stained with aniline blue for 1.5 h. Then, slides were washed with tap water and stained with Biebrich’s acid scarlet fuchsin for 10 min and subjected to 0.5% acetic acid treatment for 15 s, dehydrated using increasing concentrations of ethanol and xylol, and mounted in synthetic resin. Finally, slides were observed with the CARL ZEISS Primo Star optical microscope with magnifications of 4× and 10× for skin and 20× for lung. Images obtained from Masson’s trichrome staining of both skin and lung were analyzed using ImageJ v.2.3.0/1.53t software (U.S. National Institutes of Health, Bethesda, MD, USA) to quantify the percentage of collagen-positive areas that were determined by the collagen-positive area of total tissue area. Two independent investigators, blinded to the experimental design, semi-quantitatively classified graded the fibrosis score in Masson’s trichrome staining of skin using the fibrosis severity scale described by Gallet et al. [20]. A severity score was established from 0 to 3, where Grade 0 indicates normal structure, Grade 1 mild fibrosis with preserved structure, Grade 2 moderate fibrosis with modification of structure, and Grade 3 severe fibrosis with destruction of structure.

In addition, two independent investigators, blinded to the experimental design, semi-quantitatively classified the fibrosis score on Masson’s trichrome stain of the lung using a fibrosis score, previously reported as the Ashcroft score [21]. A score of 0 to 8 was established, where a score of 0 indicates normal lung, a score of 1 indicates minimal fibrous thickening of alveolar or bronchiolar wall, a score of 2–3 indicates moderate thickening of walls without obvious damage to lung, a score of 4–5 indicates increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses, a score of 6–7 indicates severe distortion of structure and large fibrous areas, and a score of 8 indicates total fibrous obliteration of the field.

2.6. Statistical Analysis

Experimental results were quantified using ImageJ v.2.3.0/1.53t and are presented as mean ± standard deviation (SD). To assess statistical significance, GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA) was used to perform a one-way analysis of variance (ANOVA) to compare the experimental groups. Subsequently, Tukey’s post hoc test was applied to make comparisons between all pairs of groups. A value of p < 0.05 was considered statistically significant in all analyses.

3. Results

3.1. CGA Treatment Reduces Dermal Thickness in BLM-Induced Mice Model of SSc

Skin histological changes and dermal thickness in all experimental group were analyzed using H&E staining. The results revealed significant histological alterations in the skin of the BLM group characterized by a reduction in subcutaneous fat (lipoatrophy), a reduction in skin appendages, and a marked increase in dermal thickness in BLM-treated mice compared to the CT and CGA groups. CGA treatment visibly reduced BLM-induced histological alterations, resulting in a reduction in dermal thickness, an increase in subcutaneous fat, and a recovery of skin appendages (Figure 2A). Quantitative analysis revealed that BLM group showed a significant increase in dermal thickness compared to CT (p = 0.005) and CGA (p = 0.007) groups. CGA treatment significantly (p = 0.020) reduced the BLM-induced increase in dermal thickness (Figure 2B). These findings suggest that CGA exerts positive effects on BLM-induced SSc-associated tissue alteration in mice skin.

3.2. CGA Treatment Relieves Inflammation in BLM-Induced Murine Model of SSc

Histological skin sections stained with H&E were analyzed using a blinded scoring system to evaluate the inflammation score. Results showed that the BLM-treated group of mice presented a significant increase in the inflammation score compared to CT (p < 0.001) and CGA (p < 0.001). On the other hand, in BLM + CGA group, the inflammation score was significantly reduced (p = 0.036) as compared with BLM group (Figure 2C). These findings suggest that CGA treatment has a significant anti-inflammatory effect in SSc induced by BLM in mice.

3.3. CGA Treatment Reduces Skin Fibrosis in BLM-Induced Mice Model of SSc

Masson’s trichrome staining analyzed skin changes in ECM deposition and the fibrosis score. The results showed that the skin of BLM-treated mice showed an excessive deposition of ECM compared to CT and CGA groups. CGA treatment visibly reduced BLM-induced ECM density (Figure 3A). Quantitative analysis revealed that the BLM group showed a significant increase in the percentage of positive area of ECM compared to CT (p < 0.001) and CGA (p < 0.001). CGA treatment significantly reduced (p < 0.001) the increase in the percent of positive area of ECM induced by BLM (Figure 3B).

In addition, Masson’s trichrome-stained histological skin sections were analyzed using a blinded scoring system to evaluate the fibrosis score. The results showed that the BLM-treated group had a significant increase in fibrosis score compared with CT (p < 0.001) and CGA (p < 0.001). On the other hand, in the BLM + CGA group, CGA treatment significantly reduced (p = 0.001) the fibrosis score compared with the BLM group (Figure 3C). These results suggest that CGA treatment may exert positive effects against skin fibrosis in a mice model of BLM-induced SSc.

3.4. CGA Treatment Reduces Pulmonary Fibrosis in BLM-Induced Mice Model of SSc

Pulmonary fibrosis is one of the significant complications of SSc, and its development contributes significantly to the morbidity and mortality associated with this disease. Given the clinical impact of pulmonary fibrosis in SSc, we evaluated whether CGA could exert a therapeutic effect on SSc-associated lung fibrosis in the BLM-induced mouse model, which the administration of BLM established. To assess histological changes in lung tissue, H&E staining was used. The results showed that the tissues of the CT and CGA groups had a well-defined lung architecture, with normal parenchyma and alveolar spaces, with no evidence of histological alterations. In contrast, the BLM-treated group exhibited a significant loss of alveolar structure, characterized by a thickening of the alveolar walls and a marked reduction in alveolar area compared to the CT (p < 0.001) and CGA (p < 0.001) groups. On the other hand, tissues from the BLM + CGA group showed a significant improvement in lung structure compared to the BLM-only group. Specifically, a reduction in BLM-induced structural alterations was observed, with a significant increase in the size of the alveolar spaces (p = 0.001) (Figure S1A,B). These findings suggest that CGA treatment has a protective effect against SSc-associated lung fibrosis in the BLM-induced mouse model, preserving alveolar structure.

Masson’s trichrome staining and the Ashcroft score scale were used to assess the effect of CGA on ECM deposition and fibrosis score. Masson’s trichrome staining allowed quantification of ECM deposition in lung tissues, revealing that the lungs of the BLM-treated group showed marked collagen accumulation, evidenced by a higher intensity of positive staining compared to the CT (p < 0.001) and CGA (p < 0.001) groups. In contrast, the BLM + CGA group showed a significant decrease (p < 0.001) in ECM deposition compared to the BLM group, suggesting the anti-fibrotic effect of CGA (Figure S1A–C). Additionally, the fibrosis score according to the Ashcroft scale indicated that both the CT- and CGA-only groups maintained a baseline score of 1, reflecting a normal lung structure with no signs of fibrosis. On the other hand, the BLM group had the highest fibrosis score of 6 (p < 0.001), indicating severe fibrosis. However, the BLM + CGA group showed a significant reduction (p < 0.001) in the Ashcroft score, with a value of 5, reflecting an attenuation of fibrosis compared to the BLM group (Figure S1A–D). These results indicate that CGA treatment exerts a positive effect on ECM deposition and severity of SSc-associated pulmonary fibrosis in a BLM-induced mice model.

4. Discussion

SSc is a chronic autoimmune disease characterized by progressive skin and internal organ fibrosis, vascular alterations, and persistent inflammation [1]. The inflammatory process plays a central role in the pathogenesis of SSc, contributing to fibroblast activation and fibrous ECM accumulation, ultimately leading to the disease’s fibrosis characteristic [1,5]. Although significant advances have been made in understanding the pathogenic mechanisms underlying SSc, the currently available treatment options are scarce and often fail to halt disease progression [8,9].

Our study investigated the impact of CGA, a polyphenol known for its antioxidant, anti-inflammatory, and antifibrotic properties [10,11], on the BLM-induced mice model of SSc using osmotic minipumps, one of the most widely used to study the disease due to its ability to reproduce key features of SSc such as skin and lung fibrosis and inflammation [16,17,18]. Continuous and controlled administration of bleomycin with osmotic minipumps ensures a sustained level of the agent, better mimicking the progression of fibrosis [22]. This model is relevant for the study of SSc in both sexes, as the fibrogenic response has been shown to be independent of the sex of the mice. Although SSc is more prevalent in females, studies have validated this model in both male and female mice, showing that BLM-induced fibrosis is significantly established in both cases [4,22,23]. Therefore, although the disease mainly affects females, the model is valid and useful for investigating the mechanisms of fibrosis and evaluating potential therapies.

Histological staining with H&E and Masson’s trichrome revealed that CGA administration significantly attenuates histopathological damage associated with BLM-induced skin fibrosis in mice, including increased dermal thickness, inflammation, and ECM deposition. These results suggest that CGA may mitigate some of the histological changes in the experimental model of SSc. These results align with existing evidence on the impact of CGA in regulating inflammation and fibrosis. For example, CGA has been shown to reduce histological alterations, inflammation, and fibrosis in liver tissue from a rat model of alcoholic steatohepatitis [24]. Similar results have been observed in myocardial tissue from a myocardial infarction (MI) model induced by ligation of the left anterior descending coronary artery. In this model, CGA prevented histopathological changes in the rat heart after MI, mainly through a significant reduction in inflammation and collagen fiber deposition [25]. A recent study showed that CGA can prevent histological alterations in the kidney, including glomerular basement membrane thickening, renal tubule dilatation, and fibrosis, in a murine model of diabetic kidney disease (DKD) induced by high-fat diet (HFD) and intraperitoneal injection of streptozotocin (STZ) [26]. Together, these findings propose CGA as a promising candidate for the treatment of fibrotic diseases in multiple organs including skin and lung in SSc.

Pulmonary fibrosis is one of the significant complications of SSc, contributing significantly to mortality in these patients. The development of pulmonary fibrosis in SSc is characterized by excessive ECM accumulation and pathological remodeling of lung tissue, resulting in progressive loss of respiratory function [27]. In the present study, we also evaluated the effect of CGA on SSc-associated pulmonary fibrosis in the BLM-induced mice model. Our results demonstrated that CGA administration significantly attenuated ECM deposition and the severity of pulmonary fibrosis, as evidenced by Masson trichrome staining and the Ashcroft scoring scale. Mice treated with CGA showed a remarkable reduction in the typical histological alterations observed in the BLM-only treated groups, including the loss of alveolar structure. These findings suggest that CGA exerts a positive effect on lung architecture in the context of BLM-induced fibrosis, possibly through its anti-inflammatory, antioxidant, and antifibrotic properties previously described in studies on other fibrosis models [24,25,28,29].

This study did not investigate the exact mechanism by which CGA reduces fibrosis and inflammation in SSc, encouraging the need for future research to identify the cellular and molecular processes associated with these protective effects. However, it is postulated that the antioxidant properties of CGA could be pivotal in decreasing oxidative stress and reactive oxygen species (ROS) production, which are implicated in fibrosis and inflammation [28]. In addition, the anti-inflammatory properties of CGA could inhibit the activation of immune cells and the release of proinflammatory cytokines, thus reducing the inflammatory response [24,25,28]. For example, it has been evidenced that CGA can reduce lung fibrosis by counteracting the oxidative, fibrotic, and inflammatory effects of paraquat (PQ)-induced lung toxicity in a murine model by significantly enhancing the activity of antioxidant enzymes such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) [28]. Similarly, CGA was shown to attenuate histological changes and collagen deposition in BLM-induced pulmonary fibrosis in mice, regulating critical processes involved in fibrosis development, such as endoplasmic reticulum stress, apoptosis, and cell proliferation, and this effect was attributed to the regulation of the expression of EGFR, MMP9, AKT1, BCL2, IL-1β, CHOP, and GRP78 [29,30]. Moreover, it was also shown that CGA reduced diabetes-associated pulmonary fibrosis in a streptozotocin-induced rat model. This effect was attributed to the regulation of the expression of profibrotic factors such as TGF-β1 and α-SMA, resulting in a decrease in myofibroblasts and a significant reduction in collagen deposition [31]. Furthermore, studies performed in alcoholic steatohepatitis models have revealed that CGA can offer protection to the rat liver against chronic alcohol intoxication by regulating oxidative stress. CGA counteracted the detrimental effects of chronic alcoholism by increasing SOD levels and promoting the expression of antioxidant genes such as Nrf-2, Ho-1, CAT, and GPx, while the mRNA expression of inflammatory cytokines such as Interleukin 1β (IL-1β), Interleukin-6 (IL-6), and Tumor necrosis factor-alpha (TNF-α) is decreased [24]. Moreover, in a rat model of acute MI, CGA has been shown to reduce the levels of inflammatory markers such as IL-1β, IL-6, TNF-α, and Interferon gamma (INF-γ) in plasma. In addition, CGA increased the activity of antioxidant enzymes such as SOD and CAT, which helped mitigate oxidative stress and inflammation in rat cardiac tissue [25]. Overall, the available evidence suggests that CGA has shown the ability to attenuate fibrosis in different organs through its antioxidant and anti-inflammatory properties and reduce oxidative stress, ROS production, and secretion of inflammatory cytokines.

In addition, recent studies have indicated that CGA may function as a protective agent in various experimental models. For example, pretreatment with CGA has been shown to confer protection against hepatic ischemia–reperfusion injury by mitigating oxidative stress, modulating inflammatory responses through the HMGB1/TLR-4/NF-κB signaling pathway, and reducing apoptosis [32]. Similarly, it was reported that CGA can prevent both inflammation and fibrosis in a murine model induced by CCl₄. Co-administration of CCl₄ and CGA resulted in a significant reduction in liver damage, evidenced by decreased levels of serum transaminases, type I collagen, and α-SMA expression. These effects are attributed to modulation of the TLR4/MyD88/NF-κB signaling pathway [33]. In addition, pretreatment with CGA was shown to provide significant neuroprotection against arsenite toxicity in murine models. Arsenite exposure caused a marked reduction in various brain functions, including altered neurochemicals, as well as decreased acetylcholinesterase (AChE) activity and brain-derived neurotrophic factor (BDNF) levels. Likewise, an increase in biomarkers of oxidative stress and inflammation was observed as well as a positive regulation of apoptosis-related genes such as Bax and Casp3. However, treatment with CGA was able to reverse all arsenite-induced brain alterations [34]. Available evidence suggests that CGA has protective properties in several experimental models.

Recent studies have provided evidence suggesting that CGA may have positive effects in several chronic diseases [35,36]. For example, in a phase I clinical trial of 26 patients with recurrent high-grade glioma, where patients received varying doses of CGA (2 mg/kg, 3 mg/kg, 4 mg/kg, 5.5 mg/kg, and 7 mg/kg) by intramuscular injection, it was observed that CGA was well tolerated and showed a significant benefit in patients’ overall survival; most adverse events were mild, and the most common adverse events were induration and pain at the injection sites, swelling, back pain, insomnia, hypertonia, and increased blood creatinine phosphokinase levels [36]. On the other hand, a study evaluating the supplement Altilix^®, containing CGA and derivatives, in 100 subjects with metabolic syndrome for six months showed that Altilix^® improved hepatic and cardiometabolic parameters compared to the placebo group [35]. These results suggest that CGA has therapeutic potential in a variety of diseases, both oncological and metabolic, making it a promising candidate for future clinical trials in other diseases including fibrotic diseases.

Our results provide promising evidence that CGA could be a potential therapy for lung and skin fibrosis in the context of SSc. However, further studies are required to explore the molecular mechanisms underlying its effect and to determine whether its efficacy is maintained in long-term studies and at different stages of the disease.

5. Conclusions

In conclusion, this experimental evidence shows that CGA attenuates skin fibrosis and inflammation in a mice model of BLM-induced SSc, proposing it as a promising candidate for the treatment of skin and lung damage in SSc. However, further studies are required to elucidate the cellular and molecular mechanisms underlying CGA’s therapeutic effects. These findings open new avenues for developing CGA-based therapies to potentially improve the clinical management and quality of life of patients with SSc.