Under diabetes mellitus, the administration of Galega officinalis promotes restoration of leukocyte precursors’ bone marrow pool and normalizes their proliferative activity. This plant protects the functional state of leukocytes by modulating actin cytoskeleton formation and through quantitative redistribution of leukocyte membrane glycoconjugates. Galega officinalis prevents the development of diabetes-associated oxidative stress which results in antiapoptotic activity. The normalization of leukocytes’ proliferative and functional capacity by Galega officinalis, along with its antiapoptotic and hypoglycemic effects, can improve the course of the disease and may prevent the development of complications of diabetes.
- Galega officinalis
- diabetes mellitus
- immune system
Diabetes mellitus belongs to a group of metabolic diseases accompanied by chronic inflammation and attenuation of the immune response, which subsequently contributes to the development of a number of complications . Cells that are most affected by glycemic status and insulin level are leukocytes, which play major roles in inflammation and immune responses . Constant high glucose levels result in the formation of cytotoxic compounds, leading to lower viability of peripheral blood leukocytes. This is mediated by enhanced reactive species production, activation of mitogen-activated protein kinase (MAPK) pathway, high levels of pro-inflammatory and poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) transcription factors, as well as inactivation of pro-survival pathways which altogether leads to increased apoptosis rate. The alterations in these molecular pathways are usually associated with increased leukocyte mobilization, which causes changes in their morphology and functional state [1, 3].
The multitude of diabetes mellitus complications creates the need for drugs with a wide spectrum of action, which would not only provide effective reduction of blood glucose but would also exhibit cytoprotective properties. The most commonly used anti-diabetes drug globally is metformin. Metformin shows a pleiotropic effect mediated by its hypoglycemic function, as well as inhibitory effect on oxidative stress and inflammation.
In many cases medicinal plants can be safe and effective alternatives to synthetic compounds in disease management, since they possess a unique composition of biologically active substances .
2. Effects of metformin on the immune system
Metformin (N,N-dimethylbiguanide) is an oral antihyperglycemic agent, which from a chemical point of view is a synthetic derivative of guanidine. The hypoglycemic effect of this drug is realized through the inhibition of hepatic glucose production, reducing intestinal glucose absorption and improving glucose uptake and utilization by peripheral tissues. Recent research has shed light on the pleiotropic effect of metformin, ranging from hypoglycemic function to cardio- and nephro-protection, as well as inhibitory effects on oxidative stress and inflammation [9, 10, 11].
The scientific data concerning the influence of metformin on the immune system is controversial, and its effect strongly depends on the pathology in which it is used. For example, metformin enhances antitumor immunity, but in other contexts, it can act as an anti-inflammatory or immunosuppressive agent . Metformin can suppress senescence- and cancer-related inflammation. The majority of experimental data indicates that metformin modulates leukocytes’ functional activity by activating 5′ adenosine monophosphate-activated protein kinase (AMPK). Metformin can activate AMPK in multiple cell populations, including macrophages and neutrophils [12, 13]. It has also been demonstrated that metformin inhibits innate immune response to fungal infection in an AMPK-dependent manner and lessens central nervous system inflammation .
Considering the significant modulating effect of metformin on the immune system, it is unsurprising that it has a strong effect on immunocompetent blood cells, which we discuss below.
2.1 Metformin influence on defective hematopoiesis
Studies conducted on Fanconi anemia mice showed the unique property of metformin to improve hematopoiesis by restoring hematopoietic stem cell (HSC) numbers. It also delays tumor formation, presumably via reduction of DNA damage induced by aldehydes . An important part of metformin protective effect may be conferred by aldehyde detoxification. Other mechanisms by which metformin may act to protect the cell’s DNA are reducing the activity of mitochondrial complex 1 activity, thus potentially reducing oxidative DNA damage. It is also possible that metformin can switch the metabolic balance between oxidative phosphorylation and anaerobic glycolysis and downregulate inflammatory pathways which are thought to contribute to bone marrow failure . Another study demonstrates that metformin treatment significantly inhibited the total-body irradiation-induced increase in the levels of DNA double-strand breaks and reactive oxygen species (ROS) by attenuation of NOX4 expression in HSCs. Furthermore, metformin modulates the expression of antioxidant enzymes in HSCs .
2.2 Influence of metformin on functional state of leukocytes
Many diabetic patients who receive metformin show significantly reduced neutrophil-to-lymphocyte ratio . Metformin is able to reduce hyperneutrophilia in girls with hyperinsulinemic hyperandrogenism and improves white blood cell count in women with polycystic ovary syndrome, two conditions characterized by a pronounced systemic inflammatory state . Metformin increased the number of CD8-positive tumor-infiltrating lymphocytes. Normalizing effect of metformin on the number of immunocompetent cells is associated with its ability to upregulate AMPK and as a consequence of altering energy metabolism in the cell .
Apart from metformin influence on immunocompetent cell number, this drug also can modulate their functional activity. As expected for an AMPK activator, metformin enhances cell mobility and phagocytosis, in particular in macrophages that show enhanced uptake of bacteria, synthetic beads, or apoptotic cells. The effects of AMPK activation may be due to its ability to increase availability of cell surface receptors, including αM integrin or Fc receptors or due to mechanisms that involve suppression of TLR4-associated signaling pathways. Metformin by activating AMPK regulates the process of inflammation resolution—efferocytosis and enhanced uptake of bacteria by phagocytic cells [12, 13].
Additionally, in patients with prediabetes, metformin treatment reduces the concentration of neutrophil extracellular trap (NET) components independently from glycemic control .
The normalization of phagocytosis processes and NETosis under metformin administration could suggest an effect of this drug on neutrophil activation. Indeed, metformin attenuates neutrophil activation via inhibition of mitochondrial respiratory complex I, potentially through intracellular H2O2-mediated inhibition of IκB-α degradation and thus prevention of NF-κB activation .
Immune system modulation by metformin can be realized not only by its direct influence on the immunocompetent cells but also by its ability to regulate chemokine level. Metformin causes a decrease in inflammatory markers in plasma, including soluble intercellular adhesion molecule, vascular cell adhesion molecule-1, macrophage migration inhibitory factor, C-reactive protein, IL-6, and IL-8. The anti-inflammatory action of metformin is realized by suppressing Akt, Erk1/2, and NF-B translocation. Such changes lead to blocking of pro-inflammatory signal transduction via the phosphoinositide 3 kinase pathway .
Immunosuppressive effect of metformin can be mediated by its ability to inhibit the expression of pro-inflammatory mediators (IFN-, TNF-, IL-1, IL-6, IL-17, iNOS, MMP9, and RANTES) and infiltration of immune cells, which was blocked by reducing the expression of CAMs (ICAM, VCAM, and E-selectin) on vascular cells [20, 21].
2.3 Effects of metformin on oxidative stress
Oxidative stress is the leading cause of microvascular and cardiovascular diabetes complications . Disruption of glucose metabolism causes mitochondrial superoxide overproduction in cells. An increased amount of superoxide leads to overactivity of polyol and hexosamine pathways, increased formation of AGEs (advanced glycation end products) and its receptors, and activation of protein kinase C isoforms. Altogether, this leads to the development of complications of diabetes. Simultaneously endothelial nitric oxide synthase is inactivated. Changes in the activity of these signaling pathways result in increased intracellular ROS and activation of pro-inflammatory pathways .
Considering such intimate link between diabetes and oxidative stress, anti-diabetes treatments should not only reduce blood sugar but should also possess strong antioxidant properties. Metformin satisfies both criteria; as in addition to a hypoglycemic effect, it improves the immunological parameters of patients, presumably through its antioxidant properties . In aortic endothelial cells, metformin has been shown to inhibit high glucose-dependent ROS overproduction, which was mediated by a reduction in NADPH oxidase activity and an inhibition of the respiratory chain complex 1. Another possible mechanism of metformin antioxidant properties is its ability to activate AMPK with the ensuing induction of manganese superoxide dismutase and expression of the antioxidant thioredoxin and endothelial NO synthase (eNOS). Additionally, metformin is able to reduce AGEs synthesis and the expression of their specific cell receptor called RAGE in endothelial cells [16, 23]. In addition to the abovementioned indirect mechanisms of modulation of superoxide anion intracellular production, it was found that metformin can directly scavenge ROS, in particular •OH but not O2• .
While leukocytes actively participate in ROS generation, they are highly sensitive to ROS-mediated oxidative damage. Metformin was demonstrated to have a protective effect against oxidative stress in immunocompetent cells .
Furthermore, metformin modulates the function of fMLP-activated polymorphonuclear neutrophils that quench the products of oxidative burst. Researchers hypothesized that metformin may recognize specific cell membrane sites, thereby inducing intracellular signal transduction resulting in changes in NADPH oxidase activity or in other sources of intracellular ROS . Furthermore, metformin-induced decrease in ROS levels led to a partial inhibition of lipid peroxidation in lymphocytes .
2.4 A protective role of metformin against apoptosis
Most chronic diseases, including diabetes mellitus, are accompanied by oxidative stress, which may result in apoptosis of different types of cells . Metformin has been shown to have protective role on apoptosis. The inhibition of apoptosis by metformin has been described in many cell types and under various conditions. There may be several mechanisms of apoptosis prevention. Firstly, metformin possesses good radical scavenging activity. Secondly, metformin can regulate caspase levels and induce xenobiotic phase II enzymes .
A number of authors have concluded that metformin exerts a neuroprotective effect by decreasing mitochondria-dependent apoptosis. This is achieved through the inhibition of permeability transition pore opening, blocking the release of cytochrome c and preventing subsequent cell death . A protective role of metformin against programmed cell death is likely mediated by maintaining mitochondria integrity and reducing Ca2+. This drug also lowers the expression of caspase-3, cytochrome c, and cleaved caspase-9 and reduces fragmentation of PARP-1 while increasing the expression of Bcl-2 . A similar protective effect of metformin has been described for primary rat hepatocytes. Metformin may protect against apoptosis by induction of menadione-induced heme oxygenase-1 and bcl-xl expression and the reduction of c-Jun N-terminal kinase activation [30, 31].
Given the ability of metformin to inhibit apoptosis of different cells in a variety of pathologies, it is possible to assume that it has a similar effect on immunocompetent blood cells. Indeed, it was shown that metformin markedly decreased the percentage of apoptotic cells in bone marrow cells of rats . It also reduces the activation of macrophages and inhibits the expression of COX-2 and caspase-3, thereby attenuating inflammatory responses and apoptosis .
Treatment with metformin reduces the amount of oxidant-induced DNA damage in lymphocytes. It was shown that pharmacological concentration (50 μM) of metformin could protect against prooxidant stimulus-induced DNA damage at early but not late stages. Thus, metformin likely exerts an antiapoptotic effect by reducing caspase-3 and caspase-8 activities .
3. Effects of
Galega officinalis L. on immunocompetent cells under diabetes mellitus
3.1 Component composition and hypoglycemic effect of non-alkaloid extract of
The non-alkaloid extract of
Crucially, such non-alkaloid fraction of
Blood glucose measurement evaluates current glucose concentration, which may depend on many factors (the intake and composition of food, physical activity and their intensity, the emotional state of the patient, and even the time of the day) . Thus, blood glucose concentration may not reflect the actual degree of diabetes compensation, potentially resulting in medication under- or overdosing. Therefore, today, the key indicator for treatment quality and risk of diabetes complications is the level of glycosylated hemoglobin (HbA1c) . Notably, the non-alkaloid fraction of
Sugar-reducing effect of non-alkaloid extract may be due to its complex composition [6, 36, 38]. Gas chromatography/mass spectrometry detected phytol as a component of non-alkaloid fraction of
Another notable biologically active substance from
It has been shown that quinazoline derivatives are capable to lower blood glucose level and body weight in obese animals . Notably, the non-alkaloid fraction of
High content of alpha-linolenic acid in
Based on the above statement, the sugar-lowering effect of the non-alkaloid fraction of
3.2 Regulation of bone marrow cells proliferation by
Many of diabetes complications are induced by the intensification of chronic inflammation and attenuation of the immune response. Leukocytes play major roles in inflammation and immune responses. Diabetes mellitus is accompanied by infectious and inflammatory processes, of which the most frequent are bacterial infections, which are accompanied by relapses and are difficult to treat. Changes in the proliferative activity and ratio of leukocytes and changes in their functional properties and activation of free radical oxidation are among probable causes of the propensity of patients with diabetes mellitus to infectious processes and their compromised immunological status .
Therefore, the measurement of the hypoglycemic effect is insufficient when testing the effectiveness of new antidiabetic agents. It is also necessary to evaluate the effect of potential hypoglycemic drugs on cells that are susceptible to metabolic changes in diabetes mellitus. Cells whose function is very significantly affected in the course of diabetes mellitus are white blood cells. High levels of glucose in the bloodstream cause inflammation, which primarily affects blood cells, in particular, leukocytes [47, 48].
In addition to a broad spectrum of substances with a hypoglycemic effect, the non-alkaloid fraction of
The non-alkaloid fraction of
Furthermore, the revealed influence of
3.3 Influence of
Galega officinalis on functional state of leukocytes and their antioxidant-prooxidant balance
In diabetes, abnormal immune response manifests itself not only in the imbalance in the process of leukocytes proliferation but also in the disruption of these cells’ functional activity. The main effectors of the inflammatory process are phagocytes . The effectiveness of phagocytic response is largely determined by the nature and intensity of its initial stage—chemotaxis. However, because of its complexity, chemotaxis is one of the most vulnerable forms of neutrophil reactivity . Therefore, the impairment of the functional capacity of phagocytes and other immunocytes is associated with the pathology of movement of these cells. The main mechanism that allows cell motility is actin polymerization, as it underlies in the formation of stress fibrils, lamellipodia, and filopodia .
In animals with diabetes, the non-alkaloid fraction of
F-actin is represented by two pools: (1) long microfilaments (the constitutive fraction of cytoskeleton) located near the cell membrane and reaching towards the center of the cell and (2) short microfilaments located in the submembrane cortical network. Short filaments form a very dynamic fraction, since they are the first ones to initiate polymerization of actin membrane filaments at the time of leukocytes activation . Along with F-actin high content in blood leukocytes in diabetes mellitus condition, the process of its polymerization is intensified with the formation of fraction of short actin filaments. The source of monomers for this polymerization is, to a large extent, products of cytoskeleton filaments depolymerization and, to a lesser extent, the cellular pool of monomeric actin. The increase in actin polymerization may be due to an increase in the phosphatidylinositol amount observed in diabetes mellitus . These cellular messengers may act as inhibitors of phosphorylation of actin regulatory proteins that affect the redistribution of actin filaments and reduce the content of cytoskeleton actin filaments and proportionally increase the level of actin in the short filaments and monomers fractions .
The administration of the non-alkaloid fraction of
As mentioned above, diabetes mellitus type 1 is characterized by pre-activated state of leukocytes. This state is associated with the structural and functional rearrangement of the receptor apparatus of these cells. Often, such alterations are realized through changes in the structure of surface glycoproteins that contain sialic acid . In diabetes, N-acetyl-β,D-glucosamine residues are exposed to a greater degree compared to healthy subjects, while the exposure of sialic acids linked by α2→3 and α2→6-glycoside bonds to subterminal residues (β, D-galactose, or N-acetylgalactosamine) decreases. Quantitative redistribution of glycoconjugates in leukocyte membranes leads to the modification of signaling networks involved in intercellular interactions, as well as, to the disruption of the aggregation and adhesiveness of these cells . Activation of membrane-bound neuraminidases in diabetes mellitus leads to a decrease in the total level of sialic acids on the cell membrane. Desialylation is accompanied by increased content of subterminal monosaccharide—β, D-galactose. Galactose-containing glycoproteins regulate leukocyte migration during the inflammatory process, accompanied by a dynamic rearrangement of actin cytoskeleton .
The non-alkaloid fraction of
Reduction in N-acetyl-β,D-glucosamine residue content upon
Under streptozotocin-induced diabetes, the administration of the non-alkaloid fraction of
Consequently, receptor apparatus restoration by
Thus, the non-alkaloid fraction of
Along with the decrease in the content of myeloperoxidase, the non-alkaloid fraction of the
Thus, the use of alkaloid-free
Diabetes is accompanied by neutrophil malfunction caused, to a large extent, by the development of oxidative-nitrative stress . Oxidative stress leads to the activation of immunocompetent blood cells and their aggregation and adhesion. Further, an increase in the synthesis of arachidonic acid and its metabolites, cytokines, oxygen radicals, and secretion of lysosomal enzymes take place in activated leukocytes. Altogether, it ultimately leads to the development of atherosclerosis .
Due to the presence of a large number of biologically active substances with a potential antioxidant effect in the non-alkaloid fraction of
The negative action of ROS in the body is counterbalanced by an antioxidant system, whose functioning is aimed at neutralizing free radicals, as well as repairing damages caused by them . However, in conditions of oxidative-nitrative stress, which is largely activated during diabetes, antioxidant system of blood cells cannot fully implement its protective and adaptive mechanisms. The abnormal functioning of the immune system is evident from a decrease in the superoxide dismutase, catalase, and glutathione peroxidase activity in leukocytes. Under diabetes, the non-alkaloid fraction
The protective effect of the non-alkaloid fraction of
Galega officinalis prevents leukocytes apoptosis induced by diabetes mellitus
The development of diabetes mellitus is accompanied by a significant intensification of oxidative-nitrative stress, resulting in the formation of substances with a strong proapoptotic effect. Especially sensitive to such substances are blood cells, including leukocytes. The response of immune cells to antigenic stimuli, as well as the nature, dynamics, and duration of the immune response and immunological tolerance formation are partially regulated through programmed cell death . The non-alkaloid fraction of
Other studies have shown that the use of the non-alkaloid fraction of
Another evidence for the activation of the extrinsic apoptosis pathway under diabetes is exposure on leukocytes’ immature membrane epitopes with modified sialic acid content. It takes place in response to the loss of surface membrane during cytoplasmic membrane blebbing . The administration of
On the other hand, it has been found that
Another significant confirmation of
The established antiapoptotic effect of
Metformin has become widely used in the treatment of diabetes mellitus type 2 over the last period of time. This is due to the fact that metformin, along with its hypoglycemic effect, has the potential to modulate the functioning of immunocompetent blood cells. Metformin transiently inhibits NADH:ubiquinone oxidoreductase of the mitochondrial electron transport chain. This inhibition leads to the activation of the energy sensor 5′-AMP-activated protein kinase. The activation of this enzyme results in a whole range of metabolic changes in the immunocompetent cells. Metformin is able to regulate the processes of bone marrow cell proliferation, affect the functional activity, and regulate the apoptosis processes of immunocompetent cells.
To date, practically all mechanisms of therapeutic influence of metformin are well described. Instead, the plant from which this biguanide was first obtained somewhat become underestimated. Under diabetes mellitus type 1, the non-alkaloid fraction of
Conflict of interest
The authors declare no conflict of interest.
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