MicroRNA molecules (miRs) are single-stranded, non-coding ribonucleic acid sequences of 17–25 nucleotides in length, playing an essential role in the post-transcriptional regulation of gene expression. They are created from double-stranded precursors produced by RNA polymerase II (Bartel, 2004).

Our recent studies indicate the role of miRs as micro-players of great importance in viral infections in humans and animals (Hukowska-Szematowicz et al., 2020; Ostrycharz and Hukowska-Szematowicz, 2022; Hukowska-Szematowicz et al., 2023). miRs can regulate gene expression related to apoptosis (Su et al., 2015) and oxidative stress (Banerjee et al., 2017; Fioravanti et al., 2022) during diseases, including viral ones (Hukowska-Szematowicz et al., 2020; Fioravanti et al., 2022; Ostrycharz and Hukowska-Szematowicz, 2022), and thus affect the activity status of signaling pathways (Chen, 2015; Sadri Nahand et al., 2021).

Apoptosis and oxidative balance maintain cell homeostasis and play an essential role in viral infections (Thomson, 2001; Santus et al., 2022). Many cells undergo apoptosis in response to viral infection, reducing the release of progeny virus. During viral infection, the relative expression of virus-related genes and the activation of innate antiviral response systems lead to an increase in reactive oxygen species (ROS), reactive nitrogen species (RNS), and toxic of by products energy metabolism. The imbalance between ROS and antioxidants in the body leads to oxidative stress, cell death, and tissue/organ damage in the host. ROS and the resulting changes in cellular redox status become one of the inducing factors for cell apoptosis (Valyi-Nagy and Dermody, 2005). Tissue damage related to apoptosis and oxidative stress is an essential element in the pathogenesis of infection with coronaviruses (Gain et al., 2022), herpesviruses (Sun et al., 2023), rhabdoviruses (Verma et al., 2018), and paramyxoviruses (Yang et al., 2023). So far, little is known about the molecular signatures of regulatory interactions between specific miRs and apoptosis and oxidative stress in diseases (Su et al., 2015; Banerjee et al., 2017; Klieser et al., 2019; Sadri Nahand et al., 2021; Fioravanti et al., 2022; Abolfathi et al., 2023). No studies are addressing this scientific problem in viral diseases. Understanding these interactions has diagnostic (searching for potential disease biomarkers) and therapeutic (modulating miR-dependent pathways) potential in the course of acute liver failure (ALF) and organ dysfunction in multi-organ failure (MOF) of viral etiology, which we encounter during Lagovirus europaeus infection (Tunon et al., 2003; Esteves et al., 2018; Asim et al., 2020).

Lagovirus europaeus (L. europaeus) genotypes GI.1 and GI.2 are etiological factors for Rabbit Hemorrhagic Disease (RHD), belonging to the Caliciviridae family, Lagovirus genus (Le Pendu et al., 2017). Lagovirus europaeus is a single-stranded RNA virus (Le Pendu et al., 2017). The L. europaeus GI.1 genotype includes four variants (GI.1a/GI.1b, GI.1c, and GI.1d), with the GI.1a variant being highly pathogenic and most often causing an acute form of the disease, with mortality rates of 90%–100%. However, the L. europaeus GI.2 genotype evokes per-acute, acute, subacute chronic, and subclinical forms of the disease with variable mortality of 50%–80%, depending on the strain (Rocchi and Dagleish, 2018).

Although the first RHD epidemic occurred 39 years ago, its pathogenesis mechanisms are still not fully understood (Liu et al., 1984). During RHD, many pathological changes occur in the rabbit’s organs, especially the liver (the site of virus replication), lung, spleen, and kidney (Abrantes et al., 2012). The highest titer of L. europaeus is found in the liver, lung, spleen, kidneys, trachea, and bone marrow, which are the most frequently assessed organs in the diagnostic process (Abrantes et al., 2012; Rocchi and Dagleish, 2018). The main changes in RHD are acute liver, spleen, lung, and kidney inflammation. Pathological changes include enlargement of the liver, spleen (splenomegaly), and kidneys, as well as hyperemia and pulmonary edema (results in severe failure manifesting as shortness of breath, worsening by death; Abrantes et al., 2012; Rocchi and Dagleish, 2018). In these organs, inflammatory foci rich in neutrophils and T and B lymphocytes are observed, as well as an increase in the expression of the inflammatory biomarker—miR-155-5p—in the liver, lungs, and kidneys and a decrease in the spleen (Hukowska-Szematowicz et al., 2023). It is known that systemic hemorrhagic diathesis and intravascular coagulation syndrome (DIC) can lead to death in rabbits (Trzeciak-Ryczek et al., 2015). Innate and adaptive immunity also play an essential role in the pathogenesis of RHD (Hukowska-Szematowicz, 2013), including peripheral blood leukocytes (Trzeciak-Ryczek et al., 2016, 2017; Semerjyan et al., 2019). After L. europaeus infection, the level of pro-inflammatory and anti-inflammatory cytokines increases in the serum, liver, and spleen (Teixeira et al., 2012; Trzeciak-Ryczek et al., 2016; Ostrycharz et al., 2021; O'Toole et al., 2022).

It has also been shown that apoptosis (Alonso et al., 1998; Jung et al., 2000; San-Miguel et al., 2006; Ni et al., 2009; Garcia-Lastra et al., 2010; Marques et al., 2010; Tunon et al., 2011; Chen et al., 2018), necrosis (Park et al., 1995), and oxidative stress (San-Miguel et al., 2006; Hu et al., 2020) are elements in the pathogenesis of RHD. The progression of the disease correlates with increased apoptosis not only of liver cells (Alonso et al., 1998; Jung et al., 2000; Garcia-Lastra et al., 2010; Tunon et al., 2011; Vallejo et al., 2014; Chen et al., 2018; Bebnowska et al., 2024) but also of lung, kidney, heart, and spleen cells (Alonso et al., 1998; San-Miguel et al., 2006; Ni et al., 2009; Bebnowska et al., 2023), as well as necrosis (Park et al., 1995). Further studies showed apoptosis of T and B lymphocytes in the liver, spleen, and granulocytes and lymphocytes in peripheral blood (Marques et al., 2010; Niedzwiedzka-Rystwej and Deptula, 2012; Hukowska-Szematowicz, 2013; Niedzwiedzka-Rystwej et al., 2013a,b). Hukowska-Szematowicz et al. (2020) showed that after infection of rabbits with the L. europaeus GI.1 genotype, the expression of proapoptotic miR-16-5p increased in the liver. The same studies did not show changes in the liver’s expression of miR-122-5p (potentially involved in oxidative stress) but were detected in the serum. In the same year, Hu et al. (2020) showed that oxidative stress plays a crucial role in liver damage, especially in early RHDV infection (18 h p.i.). The nuclear translocation of Keap1-NF-kB is crucial for suppressing the NFR-2-ARE pathway in hepatocytes. Upregulation of Nrf-2 protein levels in liver cell nuclei by tert-butylhydroquinone (tBHQ) delayed the death of rabbits with RHDV infection. Earlier in 2006, San-Miguel et al. (2006) showed that oxidative stress is the main apoptosis pathway in RHD.

The above findings indicate that apoptosis and oxidative stress play an essential role in the pathogenesis of RHD. However, so far [apart from the research of Hukowska-Szematowicz et al. (2020) and Ostrycharz and Hukowska-Szematowicz (2022)], nothing is known about the molecular signatures of regulatory interactions between miRs and apoptosis and oxidative stress in L. europaeus infection/pathogenesis of RHD.

Therefore, we undertook research aimed at assessing the expression of miRs and their target genes related to apoptosis and oxidative stress and assessing their potential impact on the pathways in infection with L. europaeus—two genotypes (GI.1 and GI.2) with different virulence in four tissues (liver, lungs, kidneys, and spleen). Based on in silico analysis and our previous literature data (Hukowska-Szematowicz et al., 2020; Ostrycharz and Hukowska-Szematowicz, 2022), we selected miRs/target genes in the apoptosis and oxidative stress processes. For apoptosis: miR-21 (target genes—phosphatase and tensin homolog [PTEN] and programmed cell death factor 4 [PDCD4]), miR-16b (antiapoptotic B cell lymphoma 2 [Bcl-2] and C-X-C motif chemokine ligand 10 [CXCL10]), miR-34a (p53 protein gene (p53), and silent information regulator 1 [SIRT1]). For oxidative stress: miR-122 (BTB domain and CNC homolog 1 [Bach1]); miR-132 (nuclear factor erythroid 2-related factor 2 [Nfr-2]). We also examined biomarkers of both processes in rabbits infected with L. europaeus GI.1 and GI.2 genotypes: Bax, Bax/Bcl-2 ratio, poly (ADP-ribose) polymerase (PARP), Caspase-3, and heme oxygenase-1 (HO-I) as a biomarker of oxidative stress.

Little is known about the molecular signatures of regulatory interactions between specific miRs, apoptosis, and oxidative stress in viral diseases. We were the first to examine the expression of three miRs with proapoptotic (miR-16b, miR-34a) and antiapoptotic (miR-21) effects, which regulate both the intrinsic and extrinsic apoptosis pathways, to determine their regulatory effects on target genes and their potential impact on the pathways in infection with L. europaeus. In the case of oxidative stress, we studied miR-122 and miR-132 and their target genes. To investigate apoptosis and oxidative stress as a response to the viral stimulus, we examined apoptosis biomarkers (Bax, Bax/Bcl-2 ratio, Caspase-3, PARP) and oxidative stress (HO-I).

Bcl-2 family proteins are important regulatory molecules of apoptosis. Members of this family can be divided into the antiapoptotic family, which includes Bcl-2, and the apoptotic family, which includes Bax. The ratio of antiapoptotic Bcl-2 to proapoptotic Bax contributes to the susceptibility of a given cell to apoptosis (San-Miguel et al., 2006). After infection of rabbits with L. europaeus GI.1 and GI.2 genotypes, there is an increase in the expression of the Bax gene, which promotes programmed cell death (PCD). The Bax biomarker increases as much as 12.3-fold in the liver of rabbits during L. europaeus GI.2 infection, which indicates rapid (32 h p.i.) and extensive apoptosis of hepatocytes in this organ in response to the viral stimulus. Moreover, Chen et al. (2018) reported increased mRNA Bax expression during RHDV infection in cells transfected with NSP6 at 24 and 36 h p.i. compared to the control. The same result regarding the upregulation of Bcl-2 family proteins (determined at the level of Bax and Bcl-2 mRNA) in the liver tissue and in the spleen and kidneys after infection of rabbits with the L. europaeus GI.2 genotype was obtained by Bebnowska et al. (2023, 2024). In our lung and spleen results, the Bax mRNA gene expression level indicates a lower degree of apoptosis (statistically insignificant). It is difficult to interpret the lack of changes in Bax mRNA expression in the kidneys, and it may result from negative regulation by p53. Because p53 promotes the apoptotic capacity of Bax as the main transcription factor, and since we have a reduction (22% reduction) of p53 in the kidneys during L. europaeus GI.1 infection and no changes during GI.2 infection, such a scenario is possible. Vallejo et al. (2014) stated that after infection with L. europaeus GI.1 at 30 and 36 h p.i., there is a significant inhibition of the expression of Bcl-2 and Bcl-xL, two antiapoptotic proteins involved in the intrinsic apoptosis pathway.

During apoptosis, Bax protein bound to Bcl-2 protein activates a cascade of reactions by releasing cytochrome c from mitochondria, which helps in the successive activation of Caspases and ultimately leads to cell death (Pepper et al., 1997). It has been proposed (Pepper et al., 1997) that the association and ratio of Bax to Bcl-2 determine cell survival or death after an apoptotic stimulus. High Bax levels and/or a decrease in Bcl-2 and a high Bax/Bcl-2 ratio promote apoptosis (Pepper et al., 1997). We demonstrated a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax during L. europaeus GI.1 and GI.2 genotype infection in four tissues of rabbits. We showed that the Bax/Bcl-2 ratio promotes apoptosis during infection with L. europaeus—two genotypes—and was more strongly expressed (2–3 times) during GI.2 infection. This process included, in order, the liver (535-fold and 181-fold, respectively, GI.2 and GI.1), spleen (28.3-fold and 15.8-fold, GI.2 and GI.1), and lungs (7.9-fold GI.2). In the kidney, a different effect was observed (11.2-fold and 35.6-fold, GI.2 and GI.1). Our studies are consistent with previous observations by other authors, which were carried out after infection with L. europaeus GI.1 genotype and GI.2 genotype and concerned mainly the liver (San-Miguel et al., 2006; Chen et al., 2018), as well as the kidney and spleen; however, no changes were noted in the lungs and spleen (Bebnowska et al., 2023).

Caspase-3 is the main effector of Caspase in the external and internal apoptosis pathways, acting on enzymes involved in DNA fragmentation and chromatin condensation (Duprez et al., 2009). In our studies, the recorded increase in the Caspase-3 gene at a similar level in the lungs (2.1-fold change and 2.3-fold change, respectively, GI.1 and GI.2) and in the spleen (2.1-fold change and 1.4-fold change, GI.1 and GI.2) indicates the activation of apoptosis in these organs. Garcia-Lastra et al. (2010) have shown a marked increase in Caspase-3 activity in the liver at 36 h (12.5-fold) and 48 h (12.6-fold) with RHDV-infected animals. Chen et al. (2018) found that non-structural protein 6 (NSP6)-induced Caspase-3 activity in Oryctolagus cuniculus kidney cells (RK13). However, other studies showed a significant increase in Caspase-3 mRNA in rabbit liver (Bebnowska et al., 2024) and cleaved protein Caspase-3 in lung, heart, kidney, and spleen during infection with the L. europaeus GI.2 genotype (Bebnowska et al., 2023). This methodological approach is better for measuring apoptosis levels/activity biomarkers (Caspase-3, PARP, and Bax/Bcl-2 proteins). On the other hand, in our own studies after infection of rabbits with L. europaeus GI.2, a reduction in the relative mRNA level of Caspase-3 was observed in the liver (2.2-fold reduction, 54% reduction) and in the kidneys during L. europaeus GI.1 infection (2.9-fold reduction, 66% reduction). These results are consistent with the observations of Vallejo et al. (2014), who showed that apoptosis in RHD is induced by L. europaeus GI.1 genotype in the liver occurred without significantly decreasing Caspase-3 activity at 12 h, 18 h, and 24 h p.i. In the same research (Vallejo et al., 2014), infection of rabbits with RHDV resulted in a marked increase of Caspase-3 activity only at 30 and 36 h p.i. in the liver.

PARP is widely known as an enzyme that plays a role in DNA damage detection and repair. Activation of PARP in the event of DNA damage enables poly(ADP-ribosylation) of appropriate proteins, influencing repair systems, which helps maintain genome stability (Chaitanya et al., 2010). Inhibition of the PARP protein leads to the accumulation of DNA damage, which contributes to cell death (Chaitanya et al., 2010). Caspase-3 is primarily responsible for cleaving the 116 kDa PARP-1 protein into an 85 kDa fragment, whose presence indicates that cells are undergoing apoptosis (San-Miguel et al., 2006). Our results indicate that during L. europaeus infection, there is a reduction of mRNA PARP of 76%–95% in the liver, kidneys, and spleen (no significance was found in the lungs), which may indicate the accumulation of DNA damage and the process of apoptosis in cells. In Western blot analysis, San-Miguel et al. (2006) and Vallejo et al. (2014) demonstrated that in later periods of RHDV infection (36 h and 38 h p.i.), there was marked proteolysis of PARP-1, a nuclear enzyme whose cleavage into an 85 kDA fragment by Caspase-3 confirms that cells are undergoing apoptosis. Using the same technique, other authors (Bebnowska et al., 2023) showed elevated levels of the cleaved form of PARP in the heart, kidney, and spleen during L. europaeus GI.2 genotype infection in rabbits. No significant changes were observed in the lungs. The above result indicates that in the infection of rabbits with L. europaeus—two genotypes (GI.1 and GI.2)—cell death is activated by apoptosis in four tissues—liver, lungs, kidneys, and spleen—and it was more strongly expressed after L. europaeus GI.2 infection. Apoptosis was accompanied by increased expression or reduction of apoptosis biomarkers Bax, Bax/Bcl-2 ratio, Caspase-3, and PARP.

Our study demonstrated that miR-21, involved in the regulation of the external apoptosis pathway (Su et al., 2015), has antiapoptotic effects in all tissues tested after L. europaeus infection and has an inhibitory effect on the target genes PTEN and PDCD4 and an effect on Caspase-3 which is equivalent to reduced apoptosis. The exception is the lungs, where no inhibitory mechanism is observed during infection with L. europaeus. For this event, it can be suggested that there are other mechanisms of PTEN induction (one of the most important genes of the apoptosis pathway) and PDCD4 induction that require further investigation. A similar regulation of miR-21 was observed in studies on the HBV, Chandipura virus, and hepatic stellate cells (Qiu et al., 2013; Wei et al., 2013; Fu et al., 2017; Pandey et al., 2021).

Our studies show that miR-16b, involved in regulating the intrinsic apoptosis pathway (Su et al., 2015), has a proapoptotic effect in most of the examined tissues, inhibiting Bcl-2, which increases apoptosis and increases Bax expression. This result is confirmed by previous research by Hukowska-Szematowicz et al. (2020) during L. europaeus GI.1 (GI.1a variant, strain Erfurt) infection in rabbits (2.5-fold change) and other studies (Su et al., 2015). Our current research excludes the lungs, where we do not observe changes in miR-16b and Bcl-2 expression during L. europaeus GI.1 and GI.2 genotype infection. Moreover, we have shown that miR-16b affects the increase in the chemoattractant CXCL10 in the liver, spleen, and kidneys, which may result in increased apoptosis and tissue damage. The exception is the lungs, where no change in miR-16b expression was observed, but an increase in CXCL10 expression was observed in both L. europaeus genotypes. The increase in CXCL10 expression differs from the literature data because no inhibitory mechanism via miR-16b is observed (Gao and Zhao, 2020). Perhaps in the case of infection with L. europaeus, there is an activating mechanism through miR-16b. According to one theory, miR-16b binds to the gene promoter and influences its increased transcription, which may have been reflected in our research on CXCL10. Our results differ from those recorded after HBV infection, where a downregulation of miR-16 in HepG2 cells was noted during transfection of HBV X protein. However, the results may differ due to the research model (in vitro vs. in vivo study) and the occurrence of acute and chronic forms of RHD during L. europaeus infection. A rapid course of infection may lead to faster damage to the organ and an increase in the level of miR-16 associated with apoptosis (Wu et al., 2011).

MiR-34a regulates the extrinsic apoptosis pathway, and its targets are SIRT1 and p53 (Yamakuchi et al., 2008; Hermeking, 2010; Su et al., 2015; Sharma et al., 2018; Jiang et al., 2019; Hao et al., 2021; Zhu et al., 2021). The literature describes a feedback mechanism for miR-34a in which miR-34a inhibits SIRT1, thereby increasing the expression of p53, which is known as a transcriptional activator of miR-34a (Yamakuchi et al., 2008; Jiang et al., 2019; Hao et al., 2021; Zhu et al., 2021). Our study demonstrated that miR-34a has a proapoptotic effect after infection of rabbits with L. europaeus GI.1 in the liver and after infection with L. europaeus GI.1 and GI.2 (in the spleen) and does not affect SIRT1 expression but induces p53 in these organs (GI.1 and GI.2). This induction of p53 leads to an increase in apoptosis. This increase in apoptosis may occur because miR-34 is a transcriptional target of p53, suggesting a positive feedback loop between p53 and miR-34a. A noticeable regulatory effect of miR-34a was observed in the lungs, the decrease of which induced an increase in SIRT1 and did not alter the regulation of p53. Our observations are partly consistent with those of other researchers. Jiang et al. (2019) report that the coxsackie B3 virus increases the expression of miR-34a during infection, which induces cardiomyocyte apoptosis by activating the SIRT1-p53 pathway. Studies (Sharma et al., 2018) also show that during T-lymphotropic virus-1 infection, there is an increase in miR-34a, which increases the level of p53. Additionally, researchers (Hao et al., 2021) have shown that treating infected cell lines with the p53 activator nutlin-3a leads to a further increase in miR-34a, creating a feedback loop similar to that observed during L. europaeus infection in the liver and spleen with L. europaeus GI.1 or GI.2 genotypes, respectively. Furthermore, other factors that influence organ damage increase the expression of miR-34a and p53, contributing to apoptosis and increasing the Bax/Bcl-2 ratio, similar to our studies (Sharma et al., 2018; Jiang et al., 2019; Hao et al., 2021).

Based on our previous literature review (Ostrycharz and Hukowska-Szematowicz, 2022), we indicated that miR-122 is involved in oxidative stress and regulates Bach1, thereby influencing the level of HO-I (the regulatory effect of miR-122 on the Bach1/HO-I axis;Shan et al., 2007; Qiu et al., 2010; Ostrycharz and Hukowska-Szematowicz, 2022; Wang et al., 2022). Many studies have shown that miR-122 is essential for HCV replication (Jopling et al., 2005; Jopling, 2008; Jopling et al., 2008; Amador-Canizares et al., 2018). On the other hand, literature data indicate that in the course of HCV and HBV infection in people (Ostrycharz and Hukowska-Szematowicz, 2022), there is a decrease in the expression of miR-122. It, in turn, inhibits the increase in the expression of Bach1, which in turn affects the increase in the expression of HO-I, and exerts a protective effect in this tissue microenvironment (Shan et al., 2007; Qiu et al., 2010; Ostrycharz and Hukowska-Szematowicz, 2022; Wang et al., 2022). No such effect was observed in our studies. Therefore, we propose an alternative mechanism for the action of miR-122 in the examined tissues during L. europaeus infection. Our studies indicate that miR-122 may participate in cell damage, and its increase in expression in almost all examined tissues (except for the lungs) decreases Bach1. The upregulation of miR-122, in turn, impacts the inhibition of HO-I activity, which can lead to increased tissue damage. Our results indicate that HO-I in L. europaeus infection has no protective effect. The association of the proposed pathway with clinical documentation indicates that the protective role of HO-I can only be considered in the lungs during L. europaeus GI.2 infection. In the case of miR-122, the highest expression of these molecules was recorded in the spleen, kidney, lung, and liver during L. europaeus GI.1 infection and the same tissues except the liver with GI.2 infection. The latter result is consistent with previous observations by Hukowska-Szematowicz et al. (2020) during L. europaeus GI.1 (GI.1a variant, strain Erfurt) infection in rabbits, which did not show miR-122 expression in the liver but in the serum.

The regulatory effect of miR-132 in the oxidative stress pathway in viral infection proposed in our study has yet to be studied. The involvement of miR-132 in oxidative stress has been confirmed in other diseases (Wasik et al., 2017; Zhou et al., 2020; Xu et al., 2021). Our studies suggested that miR-132 can be involved in tissue damage during L. europaeus infection. Overexpression of miR-132 during L. europaeus GI.1 and GI.2 infection may be an element of the pathogenesis of RHD. The conducted research showed increased miR-132 in the liver and spleen during L. europaeus GI.1 and GI.2 infection. Whereas in the kidney, it was noted that miR-132 expression increased only during infection with L. europaeus GI.1. The mechanism of Nrf-2 inhibition via miR-132 is observed only in the liver and kidneys. This mechanism correlates with a simultaneous decrease in HO-I expression, which may increase liver and kidney damage. Interestingly, in the spleen, despite the increase in miR-132, an increased level of Nrf-2 is observed with a simultaneous decrease in the level of HO-I. Further research is required to investigate this mechanism. Data on Nrf-2 signaling (a critical factor in oxidative defense) in viral infections are limited (Herengt et al., 2021). Evidence suggests that activation of Nfr-2 in host cells is protective during viral infections. Protection may be through either antiviral activity, inhibition of cell death to protect against excessive tissue damage, or both (Herengt et al., 2021). There was no change in miR-132 expression in the lungs after infection with L. europaeus GI.1 and GI.2. In the lungs, after the L. europaeus GI.1 infection, no HO-I changes were noted. It can be assumed that the result of these reactions is less oxidative damage in the lungs and more significant damage in the liver, spleen, and kidneys. Our results regarding the expression of Nrf-2 in response to L. europaeus infection confirm other studies (San-Miguel et al., 2006; Hu et al., 2020). Hu et al. (2020) showed that both the mRNA and protein levels of Nrf-2 were significantly reduced after RHDV infection, which shows that RHDV infection inhibits Nrf-2 activity and the antioxidant response. San-Miguel et al. (2006) showed that oxidative stress is a primary pathway for apoptosis in RHDV.

A detailed analysis of the first HO-I deficiency in a human showed that HO-I protects many tissues and organs against oxidative stress and excessive inflammatory responses by releasing many molecules with stress and antioxidant properties (Yachie, 2021). In addition, it protects against programmed cell death, and this cytoprotective effect is based on its ability to catabolize free heme and prevent cells from sensitizing to apoptosis. HO-I production is induced in vivo in selected cell types, including renal tubular epithelium, liver Kupffer cells, vascular endothelium, and monocytes/macrophages, suggesting that HO-I plays a crucial role in these cells. Data from reported cases of HO-I deficiency in humans and numerous studies in animal models suggest that HO-I plays a crucial role in various clinical conditions involving oxidative stress (Yachie, 2021). The magnitude of HO-I induction after oxidative stress and the wide distribution of this enzyme in systemic tissues, combined with the biological activity of the catalytic byproducts carbon monoxide, iron, and bilirubin, make HO-I a very attractive and interesting biomarker of oxidative stress and may play an essential role in mediating protection against liver, lung, spleen, and kidney damage (Choi and Alam, 1996). Because of the above facts, our results indicate that the reduction of HO-I mRNA at the level of 52%–69% in rabbit organs (liver, kidneys, spleen) after infection with L. europaeus GI.1 and GI.2 genotypes does not protect cells against oxidative damage but, on the contrary, may intensify, which is consistent with the results of experimental studies on various animal models (Choi and Alam, 1996; Yachie, 2021). The increase in HO-I mRNA expression (2-fold change, p = 0.02 vs. control) in the lungs during L. europaeus GI.2 infection, observed in our studies, indicates a role in protection against lung damage. Accumulating evidence suggests that oxidative stress plays a central role in the pathogenesis of many pulmonary diseases, including adult respiratory distress syndrome, emphysema, asthma, bronchopulmonary dysplasia, and interstitial pulmonary fibrosis (Choi and Alam, 1996).

Our report is the first to present the regulatory effects of miRs on apoptosis and oxidative stress genes in rabbit infection with L. europaeus—two genotypes (GI.1 and GI.2) in four tissues (liver, lungs, kidneys, and spleen). Our research provides new data that are critical for understanding the pathogenesis of Rabbit Hemorrhagic Disease caused by L. europaeus—two genotypes (GI.1 and GI.2), regarding the molecular regulation of apoptosis and oxidative stress by miRs (as two essential biological processes in viral infections). The regulatory effect of miRs indicates that, on the one hand, miRs can intensify apoptosis (miR-16b, miR-34a) in the examined organs in response to a viral stimulus and, on the other hand, inhibit (miR-21), which in both cases may be a determinant of the pathogenesis of RHD and tissue damage. Biomarkers of the Bax and Bax/Bcl-2 ratio promote more intense apoptosis after infection with the L. europaeus GI.2 genotype. Our findings demonstrate that miR-122 and miR-132 regulate oxidative stress in the pathogenesis of RHD, which is associated with tissue damage. The HO-I biomarker in the course of rabbit hemorrhagic disease indicates oxidative tissue damage. Our findings show that miR-21, miR-16b, and miR-34a regulate three apoptosis pathways. Meanwhile, miR-122 and miR-132 are involved in two oxidative stress pathways. The results of our research also have diagnostic (searching for potential disease biomarkers) and therapeutic (modulating miR-dependent pathways) potential during acute liver failure (ALF) and multi-organ failure (MOF) of viral etiology, which we encounter during L. europaeus infection.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was approved by the Local Ethical Committee for Animal Experiments in Poznań, Poland (no. 51/2022). The study was conducted in accordance with the local legislation and institutional requirements.

EO: conceptualization, data curation, investigation, methodology, software, validation, visualization, writing—original draft, and writing—review and editing. AF: investigation, resources, and writing—original draft. AK: investigation, resources, and writing—original draft. AS: investigation, resources, and writing—original draft. BHS: conceptualization, data curation, formal analysis, investigation, methodology, supervision, validation, writing—original draft, writing—review and editing, funding acquisition, and project administration.