**5. Oxidative stress and mitochondria in sepsis**

#### **5.1 Generation of reactive oxygen species (ROS)**

The internal mitochondrial membrane is a vast, impermeable structure containing enzymatic complexes of the respiratory chain (I – IV) and ATP synthase system (complex V). The transportation of electrons through respiratory chain complexes is accompanied by translocation of protons (H+ ) to the intermembrane space and development of potential difference on either side of the membrane.

That process includes development of reactive oxygen species (ROS) as a consequence of electron "escape" and mono-electron reduction of oxygen to 02**.** radical. That reaction occurs in the I and III complexes of the respiratory chain as presented in **Figure 6***.* The developing oxygen free radicals are transferred both

*Organ Damage in Sepsis: Molecular Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.98302*

**Figure 6.** *ROS production in mitochondria.*

to the mitochondrial matrix and intermembrane space. The outer mitochondrial membrane is the site of location of NADPH, which generates hydrogen peroxide (H2O2). The ROS produced in the mitochondria cause damages of the mitochondrial proteins and mitochondrial DNA (mtDNA). These damages lead to the development of a mega-channel, enabling outflow of cytochrome C into the cytosol, with consequent initiation of apoptosis process. An increased permeability of the inner mitochondrial membrane creates also a possibility of transportation of many small molecules. Mitochondrial ROS affects also many processes both under normal and pathological conditions, what as shown in **Figure 7** can modulate vital cell functions.

#### **5.2 Generation of reactive nitrogen species (RNS)**

In sepsis, besides ROS production in the mitochondria, increased synthesis of nitric oxide (NO) is also an important element of oxidative stress. NO is produced by various cells, such as: activated macrophages, neutrophils or lymphocytes. Many molecules involved in septic inflammatory process, such as: tumour necrosis factor-α (TNFα) interferon γ (IFNγ) or interleukin-1β (IL-1β) also participate in the induction of type II NO synthase (iNOS) through activation of IκB degradation and transcription of *iNOS* gene.

An adequate nitric oxide production is a prerequisite of normal vascular structure and function. Muscle cells and haematopoietic cells are an important source of NO. Numerous inflammatory mediators participate in the induction and activation of the isoform of calcium-independent nitric oxygen synthase (iNOS). In order to prevent NO overproduction during sepsis, an administration of NOS inhibitors was suggested. NOS inhibition, however, has a limited therapeutic efficacy in view of intensification of organ dysfunction, which results in a high mortality rate. This is most likely related to the double role of iNOS in sepsis [8].

In particular cases, besides NO production, iNOS catalyses also the formation of reactive nitrogen species (RNS). NO reacts with superoxide anion to form peroxynitrite anion (ONOO− ), which oxidises and nitrosylates various biological targets. Peroxynitrite can be a potential mediator of the cytotoxic effect of NO.

During sepsis, iNOS can become an important source of RNS as a consequence of enzyme decoupling. Decoupled iNOS is a source of superoxide anion, which is rapidly broken down by superoxide dismutase (SOD), and hydrogen peroxide (H2O2) is produced. Moreover, the availability of NO is reduced in view of its rapid reaction with the peroxide. During sepsis, three main factors can contribute to iNOS decoupling: suboptimal tetrahydrobiopterin (BH4) concentration, insufficient

**Figure 7.** *The role of ROS in normal and pathological conditions.*

concentration of L-arginine substrate and also increased production of asymmetric dimethylarginine (ADMA, endogenous NOS inhibitor) [8].

#### **5.3 Cellular antioxidant systems**

Cells have developed protective mechanisms counteracting mitochondrial dysfunction. The most important of them include: a system of endogenous antioxidants, dynamic changes of mitochondria and also processes of removal of damaged organelles and biogenesis of new ones. To counteract oxidative damage, mitochondria contain high concentrations of antioxidants, i.e. substances, which, when present in low concentrations, reduce the level and/or protect the substrates against their oxidative modification. ROS uptake is the function of the antioxidants. Two types of antioxidants can be distinguished: enzymatic and non-enzymatic. The important antioxidant enzymes include superoxide dismutase (SOD), catalase and glutathione peroxidise (GPx). SOD catalyses conversion of O2 . to H2O2 and oxygen. In mammalian cells two forms of SOD are present: CuZnSOD (SOD 1) and MnSOD (SOD 2). SOD 1 requires presence of copper and zinc as cofactors and is mainly present in cytosol, while SOD 2 is manganese-dependent and is mainly found in mitochondria. The hydrogen peroxide produced as a result of a reaction catalysed by SOD is then detoxicated by catalase or GPx. In the reaction catalysed by catalase, water and oxygen are produced. GPx converts H2O2 into water in a reaction, in which glutathione (GSH) is oxidised to glutathione disulfide (GSSG) and then reduced to GSH by glutathione reductase (GR). Both GPx and GR require the presence of selenium to reach their full activity.

The important non-enzymatic antioxidants include vitamins, enzymatic cofactors and many endogenous substances. The antioxidant vitamins include vitamin A, C and E, which mainly act as compounds scavenging free radicals. As mentioned above, microelements such as manganese, copper, zinc, and selenium are important elements of the antioxidant systems. Endogenous antioxidant substances include in the first place bilirubin albumin, ferritin and melatonin.

#### **5.4 Mitochondrial dynamics**

Mitochondria are dynamic organelles undergoing constant regular cycles of fusion and breakdown. The fusion of damaged mitochondria and then asymmetric breakdown are the mechanism leading to regaining of the functionality of the basic

#### *Organ Damage in Sepsis: Molecular Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.98302*

components. The damaged elements present in the organelles can be grouped, as a result of fusion, in one mitochondrium. Asymmetric breakdown leads to creation of functionally efficient organelles and of mitochondria, in which all damages are accumulated. These dysfunctional mitochondria are eliminated by autophagy [9].

Mitochondrial fusion and breakdown are strictly balanced processes. Both an uncontrolled fusion and breakdown can constitute an extreme threat to the cell function and lead to cell death. At present, the data on mitochondrial fusion and breakdown in critical conditions are scarce. An increased level of markers presenting the mitochondrial dynamics was found in *post mortem* liver biopsies but not *in vivo* in critically ill patients. In an animal model, in rabbits, which had a significant dysfunction of the hepatic and renal mitochondria, the level of mitochondrial fusion protein of the inner membrane (optic atrophy protein-1) was only significantly increased in the liver. A reduction was also observed of the concentration of mitofusin-2, involved in the fusion of the outer mitochondrial membrane. The breakdown markers remained unchanged in both organs. The results of those studies may suggest that the ability of the mitochondria to fuse and break down differs between the organs.

The elimination of dysfunctional mitochondria requires replenishing of their population through biogenesis. Mitochondrial biogenesis depends on nuclear and mitochondrial transcription systems. Peroxisome proliferator-activated receptor gamma-activator 1 alpha (PGC-1α) has been identified as the key element of the biogenesis process. It activates the nuclear respiratory factors 1 and 2 (NRF1, NRF2), which induce important transcription factors, such as mitochondrial transcription factor A (TFAM) and nuclear-encoded mitochondrial proteins – subunits of respiratory chain complexes. That complex transcription programme causes mtDNA replication and synthesis of new proteins indispensable for development of new mitochondria. That programme is extremely metabolically expensive, since it requires a huge energy expenditure.

In animal sepsis models increased levels were observed of hepatic markers of mitochondrial biogenesis: PGC-1α, NRF1 and TRAM, which was associated with regaining of metabolic activity and improvement of the clinical condition. No differences were found in the levels of hepatic and renal mitochondrial biogenesis markers in rabbits, which were in critical condition but survived, compared to the animals, which failed to survive the experiment. In patients surviving a sepsis, no changes in mitochondrial protein synthesis were observed *in vivo*, in spite of an increase of the mRNA level of mitochondrial transcription factors.

## **6. Apoptosis in sepsis**

Apoptosis plays a significant role in the pathophysiology of sepsis. The role of a potential factor involved in immunosuppression and mortality in sepsis has been ascribed to lymphocyte apoptosis. An increased apoptosis of T and B cells was observed in patients dying of sepsis. The results of clinical studies have confirmed the observations, which demonstrated a significant increase of lymphocyte apoptosis in the model of cecal ligation and puncture (CLP)-induced sepsis in mice. Although death of adaptative immune system cells, limiting thus the inflammatory reaction, may be beneficial for the body, but the presence of intense apoptosis of immune cells leads to a reduced possibility of defence during invasion of pathogens. It seems that lymphocyte apoptosis, leading to immune response impairment, can predispose to septic death. That suggestion has been confirmed by the results of studies in transgenic mice with increased expression of antiapoptotic Bcl-2 protein. In the model of CLP-induced sepsis a protective effect of Bcl-2 on T-cells has

**Figure 8.**

*Apoptosis of various cells in sepsis and its consequences for organ function.*

been demonstrated, which increased the survival. That suggests that apoptosis of immune cells plays a significant role in the development of sepsis and is of decisive importance for its possible unfavourable course. The role of a apoptosis of various cells in sepsis and its consequences for organ function is presented in **Figure 8**.

In experimental sepsis models, also an intense apoptosis has been observed of interstitial cells, such as intestinal and pulmonary epithelial cells. A defect of intestinal epithelial cells can lead to a significant impairment of their barrier function and to facilitation of bacterial translocation into blood and/or lymphatic system. That results in an increased antigen presentation and massive immune response, which have a direct effect on the survival.

Apoptosis of pulmonary epithelial cells leads to the development of acute lung injury (ALI). That pathology is directly caused not only by inflammation or trauma but also by haemorrhagic shock and multibacterial sepsis. The silencing of Fas receptors on the membranes of pulmonary epithelial cells in mice with haemorrhagic shock, leads to ALI reduction through blocking of their apoptosis and, thus, inhibition of histological remodelling of the lungs.

## **7. mTOR and autophagy in sepsis**

The mTOR pathway plays an important role in promoting sepsis progression. TLR4 activation through LPS binding increases mTOR activity. In a clinical study in 106 patients with sepsis an activation of HIF-1α and mTOR genes was observed in peripheral blood leucocytes. An injection of LPS to mice caused an activation of mTOR signalling in macrophages and led to an increase of inflammatory renal injury followed by fibrosis. An activation of mTOR pathway by LPS in rats resulted in a blood pressure reduction and heart rate acceleration and also in an increase of the level of inflammatory markers in the tissues of the kidneys, heart and blood vessels. In both mentioned animal models, an administration of rapamycin and mTOR inhibitor significantly reversed the harmful effects of LPS. The inhibition of mTOR in sepsis can be partly explained by activation of the autophagy process.

Autophagy is a degradation system, preserved in an evolutionary way in the cells, and it participates in maintaining of intracellular homeostasis. The process of autophagy includes formation of autophagosomes, fusion of autophagosome-lysosomes and development of degradation products. In sepsis, autophagy is a recognised protective adaptative mechanism limiting cell injury and apoptosis [10, 11]. Autophagy not only eliminates damaged protein aggregates and organelles, but also

#### *Organ Damage in Sepsis: Molecular Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.98302*

eliminates bacteria and pathogens present in the cytoplasm. Some special bacteria, such as *Staphylococcus aureus,* can avoid selective autophagy through activation of cell kinases of the host.

The main role of autophagy in sepsis illustrates in **Figure 9**. Autophagy promotes pathogen elimination directly by phagocytosis, regulation of antigen presentation, activation of immune cells and secretion of type I interferon. It inhibits the immune response through degradation of inflammasomes. It prevents immunosuppression through inhibition of apoptosis and elimination of damaged mitochondria. It affects the metabolism through regulation of mitochondrial functions and participation of AMPK and mTOR in the regulation of autophagy activity. That effect is, however, limited. In the case of severe sepsis, even a significant increase of autophagy fails to reverse the overwhelming inflammatory reaction. The body of evidence gathered, suggest that autophagy dynamically changes to become insufficient and non-adaptative at later stages of sepsis. Dynamic changes of autophagy during sepsis was shown in **Figure 10**. That deficit is associated with mTOR signalling regulation, while ineffectiveness of elimination of dysfunctional organelles and toxic intracellular material causes an overwhelming accumulation of dangerassociated molecular patterns (DAMPs).

Both autophagy and mTOR pathway are promoted at the initial stages of sepsis. At later sepsis stages a long-lasting drop of autophagy is observed, contributing to organ dysfunction and reduced lymphocyte count, what is important for inflammatory dysregulation, apoptosis and mitochondrial disorders. Two separate animal models demonstrated that autophagy regulation through rapamycin administration reversed the heart damage observed during sepsis. These studies have shown that mTOR is the main inhibitor of autophagy.

Autophagy has been proposed as a potential therapeutic goal, particularly during evident immunosuppression and then in the phase of sepsis, as a way of immune homeostasis restoration. It seems that autophagy promotion can be a novel

**Figure 10.**

*Dynamic changes of autophagy during sepsis.*

**Figure 9.**

and effective intervention in order to reduce organ dysfunction caused by insufficient and non-adaptative autophagy during severe sepsis.

#### **8. Non-coding RNAs in sepsis**

Non-coding RNAs are responsible for the regulation of many cell signalling pathways. The molecules include long non-coding RNA (lncRNA) and microRNA (miRNA), participating in many biological processes, such as apoptosis, mitochondrial dysfunction and innate immunity.

All cells synthesise RNA molecules of 21 to 25 nucleotide length, called miRNA. The molecules of miRNA bind to complementary sequences at 3′ ends of target mRNAs and are able to post-transcriptionally regulate the expression of many genes. They can, therefore, exert protective or harmful effects in various immune system-related disorders and affect the levels of proinflammatory cytokines: TNFα and IL-1β through signalling pathways including p38 mitogen-activated protein kinase (MAPK) and MAPK 1 phosphatase (MKP-1). Many studies have demonstrated significant differences in the expression of some miRNAs in septic patients. It was suggested, therefore, that miRNAs may serve as biomarkers in the diagnostic process or risk stratification, and even can be a therapeutic target in the treatment of sepsis.

The lncRNA family includes molecules of protein-non-coding RNAs containing about 200 nucleotides and consists of over 60,000 individually catalogued members. lncRNAs have many functions and activities such as: regulation of gene expression, changing chromatin arrangement, modification of histones and alternative gene splicing, which suggest their possible role in the pathogenesis of various diseases and disorders.

#### **8.1 MicroRNA**

MicroRNA molecules play an important role in the regulation of adaptative and innate immune responses in the pathogenesis of sepsis as presented in **Table 1**. The molecules affect the development of regulatory T-cells and also T-helper cells, which are indispensable for immune response optimisation. During sepsis the immune system of the host seems to be both in the state of immunosuppression, but also, at the same time can be in a proinflammatory state [12].

In proinflammatory state, many cytokines, including TNFα, are overexpressed. miRNAs can control TNFα production both at translation and transcription levels. miR-181 has been shown to regulate TNFα synthesis through intensification of TNFα mRNA degradation. It has been also observed that a significantly reduced miR-125b expression was accompanied by a higher TNFα expression in the monocytes of newborns after LPS stimulation. Besides that, miRNAs can also regulate the TNF pathway and mediate inflammatory reactions. It has been suggested that miR-511 is a regulator of TNF receptor protein synthesis and, thus, it affects the sensitivity of cells to TNF. An effect of miR-511 is therefore possible, protecting against TNF-dependent endotoxic shock syndrome. It has been also demonstrated that the levels of other proinflammatory cytokines, such as IL-6, are significantly increased in septic patients. Experimental studies have shown that miR-146a expression is correlated with an increased IL-6 concentration in septic patients. Many studies have demonstrated that miRNA molecules are able to regulate inflammatory reactions through their effect on the Toll-like receptor 4 (TLR4) signalling pathway. The TLR4-induced signalling activates in the first place NFκB, the key transcription factor modulating the expression of proinflammatory and

*Organ Damage in Sepsis: Molecular Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.98302*


#### **Table 1.**

*Selected miRNAs involved in human sepsis.*

immunoregulatory factors. Some miRNA molecules, such as miR-155, miR-125 and miR-146a play the main role in the negative modulation of the TLR4/NFκB inflammatory cascade, but also in innate immunity. The expression of miR-155 and also many other miRNA molecules depends on NFκB, since it has been shown that LPS stimulation of THP-1 monocytes induces the expression of miR-146a and miR146b. The miR146a molecule directly regulates the expression of TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1), which are important adaptor molecules in the TLR4 signalling pathway. miR-146a plays also a significant role in the *in vitro* tolerance of monocytes to endotoxins. That effect can be, however, reversed by miR-146a suppression. It has been recently reported that the NFκB/DICER signalling pathway inhibits TNFα synthesis through production of mature forms of miR-130 and miR-125b, which regulate TNFα mRNA. The presented miRNA effects seem particularly important in the aspect of the critical role of TLR4-induced pathway in sepsis.

#### **8.2 lncRNAs and sepsis**

The molecules of lncRNA play an important role in biological processes and their dysregulation is associated with various disorders [13]. The experimental studies presented in **Table 2***,* were conducted in order to assess the correlation between sepsis and expression of lncRNA molecules. It has been demonstrated that lncRNA expression is changed in human monocytes, cardiomyocytes and renal canalicular epithelial cells during development of sepsis or after exposure to LPS.


#### **Table 2.**

*Selected lncRNAs involved in sepsis.*

In spite of many studies on that topic, sepsis still remains a complex clinical condition, the pathophysiology of which has not been fully elucidated. Non-coding RNAs offer a chance for early diagnosis and monitoring of patients in Intensive Care Units.

#### **9. Conclusions and perspectives**

Uncontrolled inflammation, immune disorders, oxidative stress, apoptosis, mitochondrial damage and also endothelial function disorders play the key role in sepsis and organ dysfunction associated with it. Sepsis releases a series of signalling cascades, starting with recognition of the whole spectrum of pathogen-associated molecular patterns (PAMPs) and organ damage-associated molecular patterns (DAMPs). Each stage of the signalling pathways participating in the release of inflammatory mediators is extremely important for the course of the disease and can be a target point in the therapeutic strategy in septic patients. The general agreement present as yet, concerning the inflammatory response only mediated by the TLR4/NFκB pathway is not so obvious any longer. Administration of antiinflammatory drugs, including corticosteroids, or antagonists of TNFα and IL-1, failed to produce the expected effects in septic patients.

The target points in the treatment of sepsis can also be the molecules of noncoding RNA (ncRNA). Recent studies have suggested, among other findings, that the miRNA-23b molecule prevents the development of myocardial dysfunction in late sepsis. It seems therefore, that research in the field of development of drugs targeted at ncRNA molecules can be the future of antiseptic pharmacotherapy.

The new SARS-CoV-2 coronaviral disease has aroused interest in viral sepsis. Although bacteria are the predominant pathogens in sepsis, a viral sepsis cannot be disregarded. According to the literature reports, the percentage of septic patients with negative results of blood cultures for bacterial pathogens reaches 42% [14, 15]. In COVID-19 patients a septic shock and multiple organ dysfunction may develop. In view of absence of specific drugs, the current therapeutic strategies include isolation of patients, controlling of infections and maintaining normal organ

*Organ Damage in Sepsis: Molecular Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.98302*

functioning. The SARS-CoV-2 infection depends on the host's cell factors. Recent studies have demonstrated that COVID-19 virus uses the angiotensin 2-converting enzyme (ACE2) and serine protease TMPRSS2 to penetrate into host's cells. That suggests a possibility of administration of protease inhibitors as effective drugs blocking the transmission of the virus [16]. The studies on viral sepsis caused by COVID-19 could help to indicate also other target points for drugs, which would reduce the damages of the lungs and other organs.
