Neuropeptide galanin is an important member of the so-called galaninergic system. Although 4 decades have passed since its discovery (Tatemoto et al., 1983), there are still numerous biological processes where the role of galanin is not yet fully understood (Jiang and Zheng, 2022; Zhu et al., 2022). The described pleiotropic effects of galanin as a neurotransmitter include its involvement in the regulation of sleep and arousal processes, behavioral processes, anxiety, learning and memory, pain and nociception, and other processes. The galaninergic system has also been found to play an important role in many peripheral organ functions, specifically in the heart and cardiovascular system, pancreas, and gastrointestinal system, as well as in bone, connective tissue, and skin (Lang et al., 2015; Šípková et al., 2017a). The diverse effects of galanin are evident not only in typical physiological conditions but also in pathological contexts (Gopalakrishnan et al., 2021).

The pleiotropy and complexity of galanin-mediated signalization are based on the existence of three different G-protein-coupled receptors (GPCRs), namely, GalR1, GalR2, and GalR3, which transduce the biological signal through different pathways (Jiang and Zheng, 2022). In addition, new ligands with partial homology to the galanin molecule were discovered over the years: GALP (galanin-like peptide) and alarin. According to the current knowledge, only GALP is capable of activating galanin receptors, namely, GalR2/GalR3, while alarin is not, despite their partial homology. Specific receptors for alarin are not known (Fang et al., 2020; Abebe et al., 2022). The newest member of the galaninergic system is spexin, a small peptide with pleiotropic functions that can activate human GalR2 and GalR3 receptors (Behrooz et al., 2020).

There are multiple studies describing the important role of the galaninergic system in metabolism, food intake, and obesity. The hypothalamic activity of galanin through GalR1 stimulation leads to increased fat intake. Moreover, there is a capability of stimulating positive feedback, which can lead to excessive fat intake and obesity (Marcos and Coveñas, 2021). This dysregulation may be followed by glucose intolerance, leading to type 2 diabetes mellitus (T2DM) and metabolic syndrome (Fang et al., 2012). Similarly, fat intake and feeding behavior can also be modified by the activity of GALP (Takenoya et al., 2018). Finally, the role in regulating food intake, satiety status, and, subsequently, obesity risk was confirmed for spexin as well (Behrooz et al., 2020). Spexin was also shown to mitigate high-fat diet (HFD)-induced murine hepatic steatosis both in vivo and in vitro (Jasmine et al., 2016). The complex role of the galanin family peptides and their receptors is also modified by other regulatory pathways and external factors, like acute and chronic stress (Sciolino et al., 2015; Šípková et al., 2017b). The inter-species differences also should not be neglected as the results of experiments in various animal models may not provide consistent results (Kuramochi et al., 2006; Hirako et al., 2017), raising the question of extrapolation of these data to humans.

Multiple studies have confirmed the presence of galanin receptors in the hearts of different vertebrates, including laboratory mice, rats, and guinea pigs. All the types of galanin receptors were discovered in the heart tissue quite early (Wang et al., 1997a; Wang et al., 1997b), but the exact role of galaninergic signalization in the heart is still not fully understood. In guinea pigs, galanin signalization was involved in positive inotropic action and a prolonged effective refractory period (Kocic, 1998). Galaninergic signalization may also be involved in the pathophysiological response to myocardial injury; for example, the myocardial galanin content was increased after cardiac ischemia and reperfusion in rats (Ewert et al., 2008). This phenomenon could be theoretically used for the treatment of cardiac muscle ischemic injury in the future (Pisarenko et al., 2017).

Celastrol, 3-hydroxy-9β,13α-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid (Figure 1), is a pentacyclic triterpenoid (Cascão et al., 2017). It was isolated from the root of Tripterygium wilfordii, which is a plant widely used in traditional Chinese medicine with reported anti-inflammatory and anticancer effects (Lee et al., 2006). When tested as a potential therapeutic agent, it was found that celastrol is a potent leptin sensitizer and anti-obesity substance in mice (Liu et al., 2015; Ye et al., 2024). Wang et al. stated that celastrol promoted white adipose tissue browning and also protected against HFD-induced obesity by the activation of the hypothalamus–sympathetic axis (Wang et al., 2021). Kyriakou et al. found that celastrol-induced weight loss is mediated by the inhibition of leptin-negative regulator protein in the hypothalamus (Kyriakou et al., 2018). Another effect was mentioned by Abu Bakar et al., who found that celastrol interferes with mitochondrial metabolism and increases pyruvate dehydrogenase complex activity while down-regulating pyruvate dehydrogenase kinase 4 expression (Abu Bakar et al., 2022). Moreover, Fang et al. suggested that celastrol leads to weight loss by inhibiting the expression of galanin and GalR1 and GalR3 receptors in the hypothalamus of mice fed on HFD (Fang et al., 2019). In the aforementioned study (Fang et al., 2019), celastrol also led to a decrease in the plasma levels of GALP, indicating that the effect of celastrol on the galaninergic system is quite complex. Finally, celastrol has been shown to have hepatoprotective properties for liver diseases, including metabolic dysfunction-associated fatty liver disease and steatohepatitis (MASLD/MASH) (Li M. et al., 2022). The exact molecular mechanisms that are responsible for these effects remain unclear. Specifically, there are no data on the effect of celastrol on the heart and liver galaninergic system. Several authors have mentioned the possibility of using galanin or the GALP agonist/antagonist in the therapy of obesity and MASH (He et al., 2023). However, the complexity of the galaninergic system is still not fully understood, especially under specific pre-existing comorbidities. Further research is needed for the potential clinical use of galanin and celastrol in the pharmacological treatment of obesity and its related metabolic and cardiovascular diseases in humans.

Therefore, the main goal of this study was to evaluate the effect of celastrol treatment on the heart and liver galaninergic systems in the mouse model of Western-type diet-induced obesity and MASLD/MASH (Figure 1).

At the beginning of the Discussion section, it is important to stress that the aim of the presented study was not to prove or develop a mouse MASLD/MASH model, as we already did this in our previous study (Arora et al., 2023). Therefore, and to reduce the number of mice to the minimum, we used only three mice in the negative controls (STD group) as a type of background for the whole experiment, which caused some results in the positive control (WD group) not reaching statistically significant values. This may appear to be the main shortcoming of our study. Another shortcoming of our study is the fact that the impact of CEL has been exclusively studied only in male subjects, although MASLD prevalence is increasing in women with polycystic ovary syndrome and estrogen deficiency, especially after menopause, leading to higher risks of MASH and advanced fibrosis compared to men of the same age (Eng et al., 2023). However, as our main aim was to evaluate the effect of celastrol treatment (WD + CEL group) against WD/FG-induced obesity and MASLD/MASH, we preferably statistically analyzed the differences between WD and WD+CEL groups of male mice, which we discuss in the following text.

In the current study, we originally demonstrated that celastrol has beneficial effects on the Western-type diet-induced obesity and progression of MASLD to MASH in mice via reduced liver inflammation, enhanced antioxidant defense, and improved lipid and glucose metabolism in at least one set of in vivo experiments. Other studies documented similar ameliorative effects of CEL on HFD-induced obesity and fatty liver in mice (Li M. et al., 2022; Zhang et al., 2016; Ye et al., 2024). The body weight-lowering effect of CEL in our study could be at least partially explained by decreased food and energy intake. It can be interpreted by the suppression of blood GAL and GALP levels and Gal/GalR1/GalR3, NPY, and leptin expressions in the hypothalamus of obese mice, where CEL works as a leptin sensitizer (Fang et al., 2019; Liu et al., 2015; Marcos and Coveñas, 2021; Takenoya et al., 2018; Ye et al., 2024). However, we have shown that the body, liver, and fat tissue weights of CEL-treated mice on the diet were significantly lower at the end of the experimental sets, although the reduced energy end/or food intake already did not differ from that of positive controls that also had considerably reduced WD consumption, namely, in set 2. Potentially, central (hypothalamic) resistance to the long-term effect of celastrol could also develop as the development of tissue galanin resistance with persistent galanin levels in obese subjects has been described before (Marcos and Coveñas, 2021). Therefore, factors other than central anorexigenic effects could also contribute to CEL complex ameliorative properties on the body, liver, and fat tissue weights and energy metabolism (i.e., improved glucose tolerance and reduced fasting glucose levels, reduced serum total cholesterol, and decreased liver triglyceride content).

To understand the progression of metabolic disease represented by MASLD/MASH, it is crucial to assess the expression of central metabolic, oxidative status, and inflammatory genes through time. This assessment provides valuable insights into the complex impact of celastrol treatment at various stages of MASH pathogenesis in the WD/FG-induced obesity experimental model. Therefore, mRNA levels (i.e., gene expression) of the selected genes involved in MASLD/MASH progression were determined in mouse livers by employing quantitative RT-PCR: Ppargc1a (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha), Crtc2 (CREB, cAMP response element-binding protein, regulated transcription coactivator 2), Mtor (mechanistic target of rapamycin kinase), Fasn (fatty acid synthase), Acaca (acetyl-coenzyme A carboxylase alpha), Srebf1 (sterol regulatory element-binding transcription factor 1), Nrf2 (also Nfe2l2, nuclear factor, erythroid derived 2, like 2), Hmox1 (heme oxygenase 1), Sirt1 (sirtuin 1), Sod2 (superoxide dismutase 2, mitochondrial), Tnfa (tumor necrosis factor alpha), and Il1b (interleukin 1 beta). CEL produced metabolic benefits through the activation of PGC-1α, leading to enhanced energy expenditure and browning of white adipose tissue by regulating mitochondrial function and biogenesis (Fang et al., 2019; Li M. et al., 2022). Throughout our in vivo experiment, CEL upregulated Ppargc1a gene expression that was suppressed by WD/FG, resembling liver-specific-deficient PGC-1α mice with manifested hepatic steatosis (Rodgers et al., 2005). We have observed similar patterns in Crtc2 and Sirt1 gene regulation by the Western-type diet and CEL. CRTC2 is a transcriptional coactivator of CREB (cAMP response element-binding protein), which plays a role in glucose, lipid, and energy metabolism through increased PGC-1α transcription and regulation of several pathways, including the cAMP and mTOR pathways, and SRBEP1/2 (Zheng et al., 2023). There are no specific studies on Crtc2 gene expression in response to CEL, while celastrol’s impact on SIRT1 expression was described to contribute to its protective effects against fatty liver (Zhang et al., 2016). SIRT1 deactivated CRTC2 and SREBP-1c, decreasing early gluconeogenesis and lipogenesis, and activated PGC-1α, reducing adiposity and lipogenesis while promoting fatty acid oxidation (Elibol and Kilic, 2018). Reciprocally, CREB and PGC-1α can upregulate SIRT1 (Molla et al., 2020), indicating that PGC-1α, CRTC2, and SIRT1 are interconnected players in metabolic regulation, impacting processes like gluconeogenesis and lipid metabolism. Therefore, we can assume that the hepatoprotective and beneficial metabolic effects of CEL in our study can, to some extent, be mediated by the CRTC2-PGC-1α-SIRT1 pathway. Furthermore, WD/FG increased serum total cholesterol, adipose tissue weight, liver triglyceride content, and steatosis score that were accompanied by significant upregulation of lipogenic genes like Acaca and Fasn. CEL significantly decreased all these lipid metabolism parameters and both genes in the set 1 experiment, which is in accordance with other animal studies (Hu et al., 2018; Zhang et al., 2016). Later, as MASH progressed, the effect of CEL was not as evident and Acaca gene expression even increased. Fasn and Acaca are the target genes for sterol regulatory element-binding protein 1 (SREBP1) transcription factor, which is involved in glucose metabolism; fatty acid, cholesterol, and TG synthesis; and adipogenesis and is encoded by the Srebf1 gene (Soyal et al., 2015). CEL can also target SIRT1 to promote AMPK-α phosphorylation and inhibit Srebp-1c-mediated lipid synthesis against oxidative stress and inflammation (Li M. et al., 2022). Unpredictably, CEL significantly upregulated increased Srebf1 mRNA in our in vivo experiments. We assumed that this was a compensatory mechanism when CEL might inhibit the activation of SREBP1 protein through the induction of the CRTC2-PGC-1α-SIRT1 pathway as CRTC2 modulated hepatic SREBP-1c cleavage (Zhang et al., 2018). This would be confirmed by the fact that CEL decreased the expression of liver protein SREBP-1c, while the Srebf1 mRNA expression in HFD-induced steatotic liver remained unchanged (Zhang et al., 2016). CEL either did not affect or normalized significantly downregulated protein expression of maturated (active, cleaved, and nuclear) SREBP1-m in set 1 and set 2, respectively. Moreover, CEL had no additional effect on SREBP1 activity, which is calculated as the ratio of SREBP1-m to premature SREPBP1-p, which was significantly increased by feeding mice WD/FG in both sets. Therefore, we may summarize that CEL does not produce its ameliorative effect on MASH primarily through the modulation of SREBP1 activity under our in vivo experimental setting.

Due to the histological scoring system’s limitations and variability in set 1, we could not clearly demonstrate a difference between the early and late stages of the positive control MASH at weeks 16 and 20, respectively. However, at week 20 and later on (Arora et al., 2023), we observed a smaller, firmer liver with less fat, confirmed biochemically and microscopically, indicating disease progression characterized by the decreasing amount of steatotic hepatocytes, the disorganized microarchitecture, and the development of liver cirrhosis and tumors. Given the considerably inconsistent findings regarding histopathological outcomes, gene mRNA (namely, Mtor and Galr2), and oxidative stress across the experimental sets, both our research team and other scientists posit that MASLD, the transition from MASLD to MASH (i.e., early-stage MASH), and advanced MASH (i.e., later-stage MASH) represent distinct and complex phenotypes (Martin et al., 2021; Arora et al., 2023). These conditions appear to involve unique signaling pathways in the liver affected by CEL in a considerably different manner in early-stage and later-stage MASH, with CEL showing more beneficial effects in the earlier stage of MASH (i.e., set 1). For example, Nrf2, Hmox1, Sod2, and Galr2 gene expressions showed variable responses to CEL in the liver. The heightened expression of antioxidant molecules was attributed to their defensive and adaptive response against the substantial reactive oxygen species production and oxidative stress triggered by the Western diet. Moreover, the same study demonstrated that mitochondria generate less cellular oxidative stress, resulting in decreased TBARS at the stage of MASH formation (Staňková et al., 2021). Another intriguing study investigated the effects of a prolonged HFD on mRNA expression across a critical set of genes (including metabolic and antioxidant ones) in C57BL/6J mice. Although SREBP expression remained unchanged in this mouse strain, PGC-1α decreased and Nrf2 showed significant enhancement, mirroring our findings (Kim et al., 2004). Similarly, TBARS, conjugated dienes, nitrites, and gene expression of Nrf2, Hmox1, and also slightly of Sod2 were concomitantly upregulated by WD/FD with down-regulating or, more appropriately, normalizing the effect of CEL at the end of set 1. On the other hand, CEL enhanced gene expression of antioxidant molecule genes in the situation when they were induced by long-term feeding with the Western-type diet at a much lower extent accompanied by decreasing oxidative stress in the liver of positive control mice at set 2. CEL has been found to activate SOD2 and Nrf2-HO1, contributing to its hepatoprotective effects in the liver of HFD obese mice (Luo et al., 2017; Zhang et al., 2016; Li M. et al., 2022). Moreover, CEL was shown to induce HO-1 through the upregulation of Nrf2 in hepatoma cells (Tseng et al., 2017). In our study, CEL significantly decreased both the liver inflammatory score and pro-inflammatory gene expression (Tnfa and Il1b) at the end of week 16, similar to that in various other studies with mouse models of fatty liver (Zhang et al., 2016; Li M. et al., 2022). Notably, macrophage infiltration and expression of macrophage M1 biomarkers (e.g., IL-6, IL-1β, TNF-α, and iNOS mRNA) were decreased after 3 weeks of CEL treatment in the livers of C57BL/6 mice induced with HFD (Luo et al., 2017), which can, at least partially, explain our findings. Wang et al. (2020) also demonstrated potent anti-inflammatory properties of CEL that remarkably suppressed the protein levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-18, and TNF-α) while increasing the levels of anti-inflammatory cytokines (IL-10 and IL-13) in the serum and fibrotic livers of rats in a dose-dependent manner, while the lowest daily dose of 250 μg/kg CEL was not significantly effective. The relatively low CEL dose used in our study could explain why we did not achieve a significant reduction in mildly increased serum and liver TNF-α with CEL administration. Finally, our results on the anti-inflammatory effect of CEL in the early stage of MASH could be supported by the study demonstrating that transcription analysis provided a more promising means for identifying an immune profile as there are large discrepancies and lack of correlation between transcription and protein expression data when using the multiomics approach (Jacobsen et al., 2024). Taken together, we suppose that CEL is less effective on late-stage MASH in the second set of our in vivo experiments because other molecular signaling cascades and genes are involved in this stage of the disease.

The involvement of galanin in liver fibrosis and inflammation is multifaceted, with varying research findings (He et al., 2023). Galanin was found to exert its anti-inflammatory effects primarily through these receptor subtypes, particularly GalR2/3, while GalR1 is believed to be pro-inflammatory and pro-fibrogenic in the liver (Lang and Kofler, 2011; Mills et al., 2021; He et al., 2023). The hepatic expression of the Galr2 gene, which was the only detected member of the galanin family at measurable mRNA levels in the mouse liver, was also variable throughout our study. Recently, it was demonstrated that human patients with MASLD have increased levels of serum galanin and that daily 5-week-lasting treatment with CEL ameliorates HFD/high cholesterol-induced MASH in mice (He et al., 2023). Moreover, in the same study, it was shown that murine macrophages express GalR2, proposing a new role for galanin in inhibiting the pro-inflammatory phenotype of macrophages and inducing their M2-polarization (He et al., 2023). Notably, the same authors revealed that galanin can inhibit primary rat hepatic stellate cell (HSC) activation and suppress the pro-fibrogenic characteristics of HSCs by activating the GalR2 receptor, whose expression is induced in activated HSCs but undetectable in quiescent HSCs (He et al., 2016). In our in vitro experiment, celastrol did not reduce palmitic acid-induced lipotoxicity in primary hepatocytes even at the highest tested non-toxic concentration. The selective GalR2 inhibitor, M871, significantly enhanced PA-induced cytotoxicity and nitric oxidative stress alongside rather reduced lipid accumulation in hepatocytes. M871 showed its tendency to reverse the effects of CEL. These results suggest that GalR2 plays an important role in hepatoprotection independent of lipid accumulation and that, in the liver, CEL might positively affect GalR2 expression in cells other than primary (untransformed) hepatocytes (e.g., macrophages/Kupffer cells, HSCs, fibroblasts, and hepatoma cells), at least in the early stages of MASH. Spexin-mediated mitigating effects on obesity and MASLD in mice on HFD and in PA-induced HepG2 cells were eradicated by M871, proving that spexin, a known GalR2/3 agonist, produces its beneficial effect through the activation of GalR2 (Wang et al., 2022). Moreover, anti-apoptotic and anti-proliferative effects of galanin and GALP were shown to be mediated through GalR2 (Berger et al., 2004; Tofighi et al., 2008), which can explain no liver cancer development in mice under the CEL treatment. Interestingly, CEL in our study also exerted highly similar ameliorative effects as spexin in vivo, including changes in Ppargc1, Fasn, Srebf1, and Sirt1 expressions (Wang et al., 2022). Therefore, we can speculate that CEL could exert its MASH ameliorative effects through increased GalR2 gene and protein expression in the liver, namely, in set 1 of our in vivo experiments. Interestingly, fatostatin did not affect liver Galr2 gene expression, suggesting that SREBP activity has no effect on Galr2/GalR2 expression and vice versa in the liver. The liver activity of SREBP1 in our in vivo experimental settings both did not correlate with GalR2 protein expression and was not influenced by CEL.

Notably, the distribution of galanin receptor subtypes in rodents differs: GALR1 is exclusively expressed in the central and peripheral nervous systems, while GALR2 and GALR3 are widely distributed in both the central and peripheral tissues of rats (Mills et al., 2021; Waters and Krause, 2000). Previously, the expression of the Galr1 gene was detected in the brain, spinal cord, heart, and skeletal muscle; however, no mouse Galr1 mRNA was detected in the liver, kidney, testis, lung, and spleen (Wang et al., 1997b). According to RNA profiling data sets generated later by the Mouse ENCODE project, there is some extent of expression of Galr2, negligible expression of Galr3, and no expression of Gal, Galp, or Galr1 RNAs in the adult liver and heart under physiological conditions of 8-weak-adult C57BL/6J mice. Interestingly, Galp and Galr3 were revealed in embryonal liver but not in adult ones, and all three Galr RNAs were detected in adult genital fat pads, with the most pronounced one being Galr2 (Yue et al., 2014; National Library of Medicine–National Centre for Biotechnology Information, 2024). ENCODE data showed GalR2 to be the only receptor subtype in adult mouse hearts, cardiomyocytes, and H9C2 cardiomyoblasts (Boal et al., 2022). In terms of Galr2 gene expression, our observations align with the highest levels of its mRNA detected in genes related to the galanin system, both in the liver and heart of adult C57BL/6J mice, regardless of their age. Conversely, we observed an age-related increase in gene expressions of Gal, Galr1, and Galr3 (in order of decreasing quantity) in the heart ventricles, with a peak occurring between weeks 16 and 20 on the controlled STD. According to the available literature, Gal and Galr3 expressions have not been detected in the mouse heart until the completion of this manuscript. Furthermore, we had originally demonstrated that WD/FG significantly increased mRNA levels of Gal, Galr1, Galr2, and Galr3 in mouse heart ventricles compared to those in negative controls in our experiments, suggesting that other members except for GalR2 might play a role in cardiovascular physiology and pathophysiology related to metabolic diseases. We had previously described the gene and protein expressions of galanin, GALP (Škopek, 2013), and galanin receptors (GalR1, GalR2, and GalR3) in rat hearts and their modulation under stress (Škopek, 2013; Šípková et al., 2017b). Moreover, galanin’s cardioprotective and crucial role in regulating cardiac autophagy and apoptosis in hypertrophied hearts, following myocardial infarction in mice, was demonstrated (Martinelli et al., 2021). A recently published review based on the experimental data on many studies summarized that galanin plays a role in the development of insulin resistance and diabetic heart conditions; however, it also helps alleviate hyperglycemia and improves insulin sensitivity, enhances glucose utilization, and promotes mitochondrial growth in the cardiac muscle through GalR2. These findings depict GalR2 as a potential therapeutic target for treating diabetic cardiomyopathy (She et al., 2023). Moreover, genetic suppression of Galr2 in vivo (by using Galr2 knockout mice) and in vitro (by using siRNA transfection) promoted cardiac hypertrophy, fibrosis, and mitochondrial oxidative stress and eradicated the beneficial effects of galanin on mouse heart and primary cardiomyocytes, respectively. Therefore, the authors concluded that targeting GalR2 might offer novel therapeutic strategies for heart diseases (Boal et al., 2022). Stimulation of GalR2 signaling by selective galanin ligands was also presented to be involved in the attenuation of myocardial ischemic/reperfusion injury and the improvement of cardiac function in rats in vivo, which was completely reversed by M871 (Palkeeva et al., 2019; Serebryakova et al., 2023). In our in vivo experiments, CEL did not affect both Gal and Galr2 expressions that were upregulated by WD/FG, probably as a result of an adaptive mechanism to metabolic stress, thus preserving the protective effect of locally produced galanin on the heart through GalR2. Moreover, CEL was histologically demonstrated to be safe not only for the heart but also for the other major organs including the liver, spleen, lungs, kidneys, and brain of HFD-induced obese mice (Fan et al., 2022).

Contrarily to the stimulation of GalR2, which activates phospholipase C-induced signaling pathways, GALR1 and GALR3 primarily decrease cAMP-mediated signaling pathways, hence interposing the inhibitory effects of galanin (Šípková, 2017a). The data on the role of GalR1 and GalR3 in heart diseases are controversial or missing (She et al., 2023). We present evidence of GalR1 expression in the mouse heart, both at the mRNA level and at the protein level detected by immunohistochemistry. In our scenario, the failure of immunostaining for the other proteins of interest (i.e., GalR2 or GalR3) might be attributed to the fixation of heart tissue by paraformaldehyde or/and the possibility that, under the experimental conditions, the endogenous GalR2 and GalR3 adopted a unique configuration different from the immunogens used to generate the antibodies (Lu and Bartfai, 2009; Michalickova et al., 2023). In general, there is a lack of sensitive and selective antibodies capable of recognizing specific endogenous G-protein-coupled receptors like mouse GalRs (Lu and Bartfai, 2009), although previously we managed to detect immunoreactivity of all three types of receptors in rat heart sections using the completely same antibodies against mouse GalRs (Šípková et al., 2017b). Therefore, when screening the effect of different modalities on galaninergic receptors, gene expression analysis is preferably used. In our experiment, GalR1 immunoreactivity was positive in cardiomyocytes of both heart atria and ventricles and was concentrated in the intercalated discs, mimicking our previous findings in rat hearts (Šípková et al., 2017b). Intercalated discs serve as intricate structures that facilitate both mechanical and electrical connections between cardiomyocytes, showing that galanin collaborates with other transmitters to regulate cardiac function as previous research indicates that galanin, through its interaction with GalR1, decreases acetylcholine release and suppresses vagal bradycardia (Herring et al., 2012). The use of the preferable GaR1 antagonist M40 resulted in enhanced cardiac function and reduced remodeling in rats with heart failure. The potential mechanism underlying this cardioprotective effect involves upregulation of cardiac SERCA2 (sarco/endoplasmic reticulum calcium ATPase 2) and a decrease in plasma IL-6 (Chen et al., 2015). Moreover, we had previously detected both mRNA and immunoreactivity of GalR1 in endothelial cells and discussed that utilizing GalR1 antagonists in clinical settings might yield antiangiogenic effects to counteract detrimental angiogenesis (Michalickova et al., 2023).

Another review primarily outlined the favorable impacts of CEL on cardiovascular disease (CVD), derived from in vitro and in vivo preclinical research and potential underlying mechanisms, including CEL´s inhibitory effect on the central galaninergic system (Li Z. et al., 2022). As CEL in our study significantly reduced Galr1 gene expression in the heart ventricle, we can assume that it can contribute to CEL’s overall favorable metabolic effect and possibly also the cardioprotective effect. This pattern of CEL can be at least partially (via decreasing fat intake, body weight, and adiposity) related to the modulation of SREBP activity as FAT produced the same Galr1 down-regulating effect in the mouse heart. It was demonstrated in vitro that mouse Galr1 gene expression is upregulated by cAMP through a CREB-dependent mechanism (Zachariou et al., 2001) and that CREB is a co-activator of SREBP-mediated transcription of reporter genes (Oliner et al., 1996). These and our findings (i.e., enhanced Galr1 expression in hearts of obese mice on WD/FG) suggest that increasing activation of SREBPs by HFD in obese mice (Soyal et al., 2015; Zhang et al., 2016) can induce Galr1 gene expression in the heart. It seems to be favorable that CEL does not completely suppress the Galr1 gene and GalR1, as we showed, because mice with a completely knockout (KO) Galr1 gene have modified intake of only HFD (not STD) and experience glucose metabolism impairment, while Galr1 heterozygotes do not differ from wild-type mice (Zorrilla et al., 2007). Interestingly, both changes in food behaviors under challenging HFD and glucose intolerance were also described for Gal-KO mice (Ahrén et al., 2004; Adams et al., 2008), suggesting the role of galanin and GalR1 in adjusting food intake and metabolic responses to variations in dietary fat while also influencing glucose regulation in mice (Zorrilla et al., 2007).

On the contrary, the Galr3-KO (backcrossed on the C57BL/6 background) mouse strain displayed physiological breeding and physical development alongside a similar trend (n.s.) of higher plasma TG and cholesterol levels compared with age-matched wild-type mice. Notably, male Galr3-KO mice exhibited an anxiety-like phenotype and reduced social affiliation (Brunner et al., 2014). However, the role of GalR3 in CVDs remains unclear as the expression of Galr3 is generally very low (i.e., as in our study) or missing depending on the age, tissue, and species, as we discussed above. Furthermore, CEL significantly increased mouse heart Galr3 expression only when it was not upregulated in positive controls. On the other hand, FAT significantly downregulated WD/FG-enhanced Galr3 mRNA, like in the cases of Gal, Galr1, and Galr2 genes, indicating the role of SREBP and fat metabolism in the gene expression of members of the galaninergic system in the heart of obese mice. To the best of our knowledge, this significant downregulating effect of FAT on the galaninergic system in the heart has not yet been described anywhere. However, both SREBP1 and GalR1/GalR2 played pivotal roles within the adipogenic signaling pathways induced by the HFD in mice. Notably, cinchonine, a natural compound, exerted simultaneous inhibitory effects on these pathways (i.e., it significantly downregulated gene expression of Srepbf1, Galr1, and Galr2 in fat tissue), resulting in reduced adipogenesis and mitigated obesity (Jung et al., 2012). However, the specific regulatory relationship between SREBPs and the galaninergic system remains unclear based on the current research. Therefore, more extensive studies are required to elaborate on the SREBP–galaninergic system interactions in various organs (including the heart) and species. Taken together, we can assume that the peripheral effects of CEL on the heart are produced mainly by decreasing the expression of Galr1 in WD/FG-induced obese mice. It can be supported by the fact that CEL was likewise demonstrated to both effectively inhibit fat intake and promote weight loss by downregulating the expression of galanin and its receptors (specifically GalR 1 and 3) in the mouse hypothalamus during HFD conditions (Fang et al., 2019).

According to our results, CEL may have a beneficial effect on the galaninergic system modulation in the heart and liver of obese male mice on the Western-type atherogenic diet. Along with reducing the fat intake, weight gain, and amelioration of MASLD to MASH progression in mice, CEL also affected the expression of galanin and its receptors in the heart ventricles and liver lobes, which may be involved in the regulation of energy metabolism, oxidative stress, and inflammation. Therefore, CEL may be a potential therapeutic agent for these metabolic dysfunction-associated disorders in humans. However, the exact mechanisms and implications of celastrol’s action on the galaninergic system are not fully understood and require further investigation. Future research should also evaluate its impact on female animal models of MASLD/MASH, particularly those with deficits or alterations in sex hormone levels, highlighting the need for personalized treatment approaches for women.