The findings of this study demonstrated the anti-inflammatory, antioxidative, and liver-protective effects of UDCA and CDCA in the LPS-induced model of inflammation in rats. This is based on the ability of these bile acids to reduce serum levels of pro-inflammatory cytokines, soluble ICAM-1, and liver enzymes, decrease prooxidative markers, increase antioxidative enzymatic activity, normalize serum lipid status, and to reduce the NF-κB expression in hepatocytes and mitigate liver injury.
The application of LPS triggers acute systemic inflammation by activating the immune system via its binding to a receptor complex composed of CD14, TLR4, and myeloid differentiation factor-2 (MD-2) [32]. The signaling through TLR4 upon binding of LPS is transmitted by two cascades: the first one involves adaptor proteins myeloid differentiation primary response gene 88 (MyD88) and Toll/IL-1 receptor (TIR) adaptor protein (TIRAP) in the plasma membrane; the second one engages adaptor proteins TIR domain-containing adaptor-inducing interferon-beta (TRIF) and TRIF-related adaptor molecule (TRAM) in early endosomes after endocytosis of the receptor complex. The MyD88-dependent pathway includes the activation of NF-κB, by dissociating its inhibitory subunit, and subsequent translocation of the activated form to the nucleus where it activates numerous genes coding pro-inflammatory cytokines, chemokines, growth factors, and immune activation biomolecules. The TLR4/TRIF pathway induces the phosphorylation of interferon-regulatory factor-3 (IRF-3) followed by the activation of genes coding IFN-β. These signaling pathways are competitive and mutually exclusive [33]. An early effect of LPS on diverse cell types, including neutrophils, macrophages, mast cells, platelets, and endothelial cells is characterized by the production of IL-1 and TNF-α. These cytokines play a crucial role in promoting the pro-inflammatory state, triggering the release of additional cytokines like IL-6, IL-8, GM-CSF, INF-γ, and other cytokines of both innate and adaptive immunity [34]. The results from our previous study clearly showed that a non-lethal dose of LPS induced a significant increase in concentrations of TNF-α, IL-1β, and IL-6 within the first 2 hours, followed by a slow and sustained decrease in subsequent hours [35]. These results are confirmed in this study and extended to GM-CSF, IL-2, and IFN-γ.
Numerous investigations showed that UDCA has an anti-inflammatory effect [11–13, 21], suggesting the benefits of this bile acid therapy in acute inflammatory response. A similar effect was published for CDCA as well [22–24, 36], but some studies suggested its pro-inflammatory role in promoting liver injury [25, 26]. Our results demonstrated that both bile acids can attenuate the production of IL-1β, TNF-α, IL-6, GM-CSF, and IFNγ induced by the LPS administration, suggesting its potential beneficial effect in suppressing systemic inflammation. At the same time, these results suggested that NF-κB could be a key target of their effect. However, other targets could be also involved such as Akt, ERK, JNK, and p38 [21]. Some differences observed (suppression of IFN-γ by CDCA or suppression of IL-6 by UDCA) could be the consequence of interindividual variability between the groups or different affinity of these bile acids to their receptors as shown for FXR and TGR5 [24]. Although UDCA and CDCA have different structures, they can act on different receptors and signaling molecules in a given immune cell, and therefore, the outcome may be the result of competition among different receptors and generated signals [37, 38]. In this context, CDCA has been shown to inhibit autophagy in an FXR-dependent mechanism, whereas UDCA stimulates the formation of autophagosomes independently of FXR and enhances autophagic flux [39]. Neither CDCA nor UDCA exerted any significant effects on the production of pro-inflammatory cytokines when applied alone, suggesting that these bile acids could be safe under physiological conditions.
Hepatomegaly, often characterized by an increase in liver weight, is a common observation in sepsis, and it can be attributed to various pathophysiological mechanisms. The liver weight-to-body weight ratio serves as an indicator of hepatomegaly and functions as a marker for liver disease [40]. UDCA, as a secondary bile acid, is known for its reduced cytotoxicity compared to primary bile acids synthesized in the liver [41]. The anti-inflammatory and antioxidant properties of UDCA and CDCA play a role in preserving liver function, reducing hepatomegaly, and enhancing the overall prognosis of sepsis [42]. The results of this study showed an increase in LW and LW/BW ratio in the LPS-treated group, indicating that LPS caused liver enlargement. In the current study, UDCA and CDCA pretreatment decreased the LW/BW ratio, and CDCA also reduced the LW, thereby preventing LPS-induced liver enlargement.
The presence of LPS in the cell wall of Gram-negative bacteria initiates an inflammatory response, leading to significant alterations in glucose metabolism. Hyperglycaemia is commonly detected in bacterial infections and serves as an indicator of unfavorable clinical prognosis [43]. Bile acids possess hormone-like properties in regulating both, lipid and glucose metabolism. The administration of UDCA resulted in reduced glucose levels, elevated serum GLP-1 levels, and relief from hyperinsulinemia [44]. However, our results showed that UDCA and CDCA did not affect the glucose levels in rats treated with LPS.
Sepsis is frequently linked with rhabdomyolysis, and Gram-positive bacterial pathogens are commonly cited as the leading cause of sepsis-induced rhabdomyolysis. The key clinical indicator in the development of rhabdomyolysis is the elevation of creatine kinase (CK) levels [45]. In this study, we observed an increase in CK levels in the LPS group, and this increase was mitigated by the administration of UDCA.
In cases of toxic injury, hepatocyte necrosis leads to the release of enzymes into the bloodstream. AST and ALT are the most frequently utilized indicators of hepatocyte damage. In comparison to AST and ALT, lactate dehydrogenase (LDH) is a less specific marker for hepatocyte injury [46]. The AST, ALT, and LDH levels increased after LPS injection [47]. Our results showed a significant increase in AST and LDH levels in the LPS-treated group and a decrease of LDH in the UDCA-treated group, suggesting that UDCA mitigates the LPS-induced changes in LDH activity.
Limited-scale investigations have indicated that increased troponin levels can identify septic patients with an increased risk of mortality. Furthermore, patients with elevated troponin levels exhibited a higher mortality rate [48]. Individuals affected by sepsis often display increased levels of cardiac troponin I, even in the absence of coronary artery disease [49]. Our study demonstrated a noteworthy rise in hsTnI levels in rats treated with LPS, which were mitigated by UDCA. However, CDCA did not result in a reduction of CK, LDH, and hsTnI levels. The rationale behind these results could be found in the structural composition of bile acids, where the cytotoxicity is influenced by their specific molecular arrangements. Notably, UDCA and CDCA represent structurally distinct bile acids. UDCA, resulting from the dehydroxylation of free CDCA, emerges as the least toxic among them.
Homocysteine has been identified as a pro-inflammatory compound that can stimulate the production of specific cytokines, potentially contributing to the development of cardiovascular disease [50]. Elevated levels of homocysteine in sepsis have been linked to higher mortality rates. Homocysteine plays a significant role in septic patients due to its pro-inflammatory and procoagulant effects [51]. The favorable impact of UDCA on dyslipidemia and cardiovascular disease risk can potentially be explained by several antioxidant mechanisms. UDCA treatment appears to act against oxidative damage dependent on iron and hydroxyl radicals, inhibits the products of lipid peroxidation, and prevents the oxidative stress induced by reactive oxygen species through the activation of the PI3K/Akt/Nrf2 pathway in hepatocytes [52]. Recent research has shown a notable increase in oxidative stress products with the thickening of arterial intima and an elevated peroxidative glutathione redox status has been associated with the progression of atherosclerosis [53]. The current investigation showed that LPS led to an increase in homocysteine levels, which was prevented by UDCA and CDCA in rats treated with LPS.
The lipid profile was assessed by analyzing TC, HDL, LDL, and TG levels. Rats treated with LPS displayed elevated levels of TC, LDL, and TG. These findings are in alignment with those of Brigatto et al [54], except for the increase in HDL concentration. UDCA is known for its potential to ameliorate dyslipidemia and reduce the risk of atherosclerotic cardiovascular disease due to its antioxidant properties [52]. In the current investigation, both UDCA and CDCA prevented the impairment of the lipid profile induced by LPS.
Endothelial cells may also have different immune functions and play a pivotal role in the systemic response to bacterial infections, working to limit their spread. When exposed to pathogens and microbial toxins, the responses of endothelial cells are diverse, heterogeneous, and multifaceted. During sepsis, endothelial cells transform into a proapoptotic, pro-inflammatory, proadhesive, and procoagulant phenotype. Additionally, damage to the glycocalyx and impaired vascular tone disrupt microcirculatory blood flow, leading to organ damage and the potential for organ failure [55]. In endothelial cells, UDCA counters endothelial dysfunction by inhibiting endoplasmic reticulum stress, reducing the expression of the receptor for advanced glycation end products, dampening the inflammatory response (including NF-κB activation), and suppressing the production of ROS. These effects are particularly valuable under hyperglycemic conditions, which are also present in sepsis [17]. Our findings indicate that UDCA and CDCA pretreatment reduced the level of ICAM thus ameliorating endothelial disfunction in the context of LPS-induced endotoxemia.
The presence of oxidative stress in LPS-induced endotoxemia has been already documented [56–58]. UDCA mitigates oxidative stress by acting as a molecule that scavenges ROS and reinforces the endogenous antioxidant defense [15]. Our study revealed a reduction in intracellular ROS levels, which had been elevated due to LPS, following treatment with UDCA and CDCA. We analyzed prooxidative markers and the antioxidative potential of UDCA and CDCA. The findings confirmed that LPS induced a significant level of oxidative stress, as indicated by the increased levels of H2O2 and O2−. In the current study, UDCA decreased the levels of H2O2 and O2−, while CDCA solely decreased the H2O2 levels. We assessed antioxidative defense by examining the activity of CAT, SOD, and GSH levels. The activity of CAT and SOD, as well as the level of GSH, were found to be decreased in LPS-induced endotoxemia, aligning with the results of several previous studies [20, 59]. Notably, both UDCA and CDCA significantly elevated the activity of CAT and GSH levels, thus increasing their antioxidative effects in the context of endotoxemia.
LPS is widely recognized as a stimulator of MAPKs, specifically the ERK, JNK, and p38 signaling pathways, which play crucial roles in regulating inflammatory responses. These pathways undergo phosphorylation in response to LPS. Additionally, NF-κB serves as a key regulator in pro-inflammatory signaling pathways. Initially present in the cytoplasm in an inactive form along with the inhibitory protein IκBα, NF-κB undergoes translocation to the nucleus for downstream inflammatory processes when IκBα is phosphorylated by inflammatory stimuli [21]. UDCA inhibits NF-κB by functionally modulating both the glucocorticoid receptor and the transcription processes dependent on NF-κB. Given the genes and proteins associated with NF-kB-dependent gene and the inactivator for NF-κB (IκBα), the reported observations propose that UDCA contributes to suppressing the production of pro-inflammatory cytokines and NO by deactivating NF-κB [60]. Certain studies have suggested that UDCA has the potential to hinder the activation of NF-κB and mitigate the phosphorylation of ERK, JNK, and p38 signals associated with inflammatory pathways [61]. A similar effect was published for CDCA as well [62]. In the present study, exposure to LPS resulted in a notable elevation in NF-κB immunoreactivity within liver tissue, and UDCA and CDCA exhibited a significant decrease in the LPS-induced overexpression of NF-κB in the liver tissue.
Pathohistological analysis revealed the normal homogenous structure of liver tissue in the control group, unlike the LPS-treated group where the liver lobule architecture, vascular congestion, hemorrhage, extravasation, determination of hepatocyte necrosis, and the presence of inflammatory cells were observed. Our results showed that the UDCA and CDCA protect liver cells from damage caused by LPS. At the same time, the administration of LPS alone leads to a significant liver injury, with changes in the structure of liver cells and sinusoids.
In conclusion, our experimental study indicates that prior administration of UDCA and CDCA offers advantageous outcomes in mitigating LPS-induced endotoxemia in rats. Our findings suggest that UDCA and CDCA, which are structurally distinct bile acids and exert slightly different effects, could both potentially play a role in preventing severe conditions associated with endotoxemia. This hypothesis is based on the findings that pretreatment with UDCA and CDCA reduces the levels of serum pro-inflammatory cytokines and NF-κB expression in hepatocytes, counteracts alterations in LW/BW ratio, prevents liver injury, improves lipid profiles, reduces oxidative stress and enhance antioxidative enzyme capacity.