Anti-Opisthorchis felineus effects of artemisinin derivatives: An in vitro study.

BACKGROUND: The drug of choice for the treatment of opisthorchiasis caused by trematodes Opisthorchis viverrini and O. felineus is praziquantel (PZQ), but there is a constant search for new anthelmintics, including those of plant origin. Positive results on the use of artemisinin derivatives against O. viverrini opisthorchiasis have been shown previously, but the effect of these compounds on O. felineus has not been studied. Therefore, here, a comparative analysis of anthelmintic properties of artemisinin derivatives (artesunate [AS], artemether [AM], and dihydroartemisinin [DHA]) was carried out in vitro in relation to PZQ. Experiments were performed on newly excysted metacercariae (NEMs) and adult flukes of O. felineus. RESULTS: Dose- and time-dependent effects of artemisinin derivatives and of PZQ were assessed in terms of motility and mortality of both NEMs and adult flukes. The most pronounced anthelmintic action was exerted by DHA, whose half-maximal inhibitory concentrations (IC50) of 1.9 (NEMs) and 2.02 µg/mL (adult flukes) were lower than those of PZQ (0.56 and 0.25 µg/mL, respectively). In contrast to PZQ, the effects of DHA and AS were similar when we compared the two developmental stages of O. felineus (NEMs and adult flukes). In addition, AM, AS, and especially DHA at doses of 100 µg/mL disrupted tegument integrity in adult flukes, which was not observed with PZQ. CONCLUSIONS: Artemisinin derivatives (AS, AM, and DHA) have good anthelmintic efficacy against the trematode O. felineus, and the action of these substances is comparable to (and sometimes better than) the effects of PZQ.

Curcumin and Its Supramolecular Complex with Disodium Glycyrrhizinate as Potential Drugs for the Liver Fluke Infection Caused by Opisthorchis felineus.

Opisthorchiosis is a parasitic liver disease found in mammals that is widespread throughout the world and causes systemic inflammation. Praziquantel remains the drug of choice for the treatment of opisthorchiosis, despite its many adverse effects. An anthelmintic effect is attributed to the main curcuminoid of Curcuma longa L. roots-curcumin (Cur)-along with many other therapeutic properties. To overcome the poor solubility of curcumin in water, a micellar complex of curcumin with the disodium salt of glycyrrhizic acid (Cur:Na2GA, molar ratio 1:1) was prepared via solid-phase mechanical processing. In vitro experiments revealed a noticeable immobilizing effect of curcumin and of Cur:Na2GA on mature and juvenile Opisthorchis felineus individuals. In vivo experiments showed that curcumin (50 mg/kg) had an anthelmintic effect after 30 days of administration to O. felineus-infected hamsters, but the effect was weaker than that of a single administration of praziquantel (400 mg/kg). Cur:Na2GA (50 mg/kg, 30 days), which contains less free curcumin, did not exert this action. The complex, just as free curcumin or better, activated the expression of bile acid synthesis genes (Cyp7A1, Fxr, and Rxra), which was suppressed by O. felineus infection and by praziquantel. Curcumin reduced the rate of inflammatory infiltration, whereas Cur:Na2GA reduced periductal fibrosis. Immunohistochemically, a decrease in liver inflammation markers was found, which is determined by calculating the numbers of tumor-necrosis-factor-positive cells during the curcumin treatment and of kynurenine-3-monooxygenase-positive cells during the Cur:Na2GA treatment. A biochemical blood test revealed a normalizing effect of Cur:Na2GA (comparable to that of curcumin) on lipid metabolism. We believe that the further development and investigation of therapeutics based on curcuminoids in relation Opisthorchis felineus and other trematode infections will be useful for clinical practice and veterinary medicine.

Prolonged liver fluke infection combined with alcoholization: An experimental mouse model.

Liver fluke infections disrupt the bile-excreting function of the human liver. Worldwide, excessive alcohol consumption also leads primarily to liver diseases. Our aim was to comprehensively assess the liver state in mice in parallel with the characterization of inflammation when the two adverse factors were combined. C57BL/6 mice were used for the experimental modeling; half of them beforehand were gradually accustomed to consumption of increasing doses of ethanol (from 5% to 20%). Then, half of the animals in each subgroup was infected with Opisthorchis felineus helminths. Finally, the infected (OF), 20% ethanol-consuming (Eth), and subjected to both factors (Eth + OF) mice were compared with no-treatment control. In OF and especially Eth + OF mice, relative liver weight was greater, activities of alanine aminotransferase and aspartate aminotransferase were higher, and bile ducts were considerably enlarged. Eth + OF mice contained noticeably more helminths in the liver than OF mice did. Massive cholangiofibrosis and periductal fibrosis were noted in the liver of the infected mice, especially Eth + OF ones. The liver fluke infection caused inflammatory infiltration and bile duct proliferation. Splenomegaly due to structural changes in the spleen as well as increased levels of interleukin 6 and leukocyte and monocyte counts in the blood reflected substantial inflammation in Eth + OF mice. Thus, in the proposed experimental model, it is shown that a double hit to the liver, i.e., the combination of O. felineus infection with prolonged alcoholization, can be detrimental to both the liver and whole body.

Behavioral effects and inflammatory markers in the brain and periphery after repeated social defeat stress burdened by Opisthorchis felineus infection in mice.

The combination of 4-week repeated social defeat stress (RSDS) and Opisthorchis felineus infection was modeled in C57BL/6 mice. Various parameters were compared between three experimental groups of male mice (SS: mice subjected to RSDS, OF: mice infected with O. felineus, and OF + SS: mice subjected to both adverse factors) and behavior-tested and intact (INT) controls. The combination caused liver hypertrophy and increased the blood level of proinflammatory cytokine interleukin 6 and proteolytic activity of cathepsin B in the hippocampus. Meanwhile, hypertrophy of the spleen and of adrenal glands was noticeable. Anxious behavior in the elevated plus-maze test was predominantly due to the infection, with synergistic effects of an interaction of the two adverse factors on multiple parameters in OF + SS mice. Depression-like behavior in the forced swimming test was caused only by RSDS and was equally pronounced in SS mice and OF + SS mice. Helminths attenuated the activities of cathepsin B in the liver and hypothalamus (which were high in SS mice) and increased cathepsin L activity in the liver. The highest blood level of corticosterone was seen in SS mice but was decreased to control levels by the trematode infection. OF mice had the lowest level of corticosterone, comparable to that in INT mice. Thus, the first data were obtained on the ability of O. felineus helminths-even at the immature stage-to modulate the effects of RSDS, thereby affecting functional connections of the host, namely "helminths â†’ liver↔brain axis."

Effects of Three-time Administration of a Supramolecular Complex of Praziquantel with Disodium Glycyrrhizinate on Trematode Opisthorchis felineus in Hamsters.

BACKGROUND: Praziquantel (PZQ) is the most commonly used anthelmintic drug for treating trematodiases. It was shown here that PZQ in complex with disodium glycyrrhizinate (PZQ-Na2GA, in the 1:10 ratio) has higher bioavailability than PZQ alone. Our aim was to determine the effects of three-time administration of PZQ-Na2GA in an experimental opisthorchiasis felinea model. METHODS: The PZQ-Na2GA complex (1:10) at a 400 mg/kg dose (meaning 36.4 mg/kg PZQ) was administered to Opisthorchis felineus-infected hamsters three times under a "9:00 am-6:00 pm-9:00 am" regimen (PZQ-Na2GA × 3). Effects of treatment were assessed as a reduction of helminth load in the hamsters and as changes in physiological, hematological, and blood biochemical parameters. The helminths extracted from the liver of the hamsters that received PZQ-Na2GA thrice were assayed for sensitivity to PZQ in vitro. RESULTS: PZQ-Na2GA × 3 reduced the number of O. felineus helminths in the liver by 87%, which is 30% better than a previously reported effect of one-time administration of the complex. Meanwhile, relative weights of the liver and thymus diminished, and some hematological parameters improved. The helminths extracted from the hamsters 1 month after the PZQ-Na2GA × 3 treatment showed elevated sensitivity to PZQ, as determined in vitro. CONCLUSION: Compared with previously published data on the effectiveness of various drugs in experimental opisthorchiasis felinea, PZQ-Na2GA × 3 exerts the most potent anthelmintic effect. In addition, PZQ-Na2GA × 3 improves physiological status of O. felineus-infected hamsters and sensitizes the surviving parasites to subsequent PZQ treatment.

The pathogenic potential of the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice.

Parasitic food-borne diseases and chronic social stress are frequent attributes of day-to-day human life. Therefore, our aim was to model the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice. Histological examination of the liver revealed inflammation sites, pronounced periductal fibrosis, and cholangiofibrosis together with proliferation of bile ducts and hepatocyte dystrophy in the infected mice, especially in the stress-exposed ones. Simultaneously with liver pathology, we detected significant structural changes in the cerebral cortex. Immunohistochemical analysis of the hippocampus indicated the highest increase in numerical density of Iba 1-, IL-6-, iNOS-, and Arg1-positive cells in mice simultaneously subjected to the two adverse factors. The number of GFAP-positive cells rose during repeated social defeat stress, most strongly in the mice subjected to both infection and stress. Real-time PCR analysis showed that the expression of genes Aif1 and Il6 differed among the analysed brain regions (hippocampus, hypothalamus, and frontal cortex) and depended on the adverse factors applied. In addition, among the brain regions, there was no consistent increase or decrease in these parameters when the two adverse treatments were combined: (i) in the hippocampus, there was upregulation of Aif1 and no change in Il6 expression; (ii) in the hypothalamus, expression levels of Aif1 and Il6 were not different from controls; and (iii) in the frontal cortex, Aif1 expression did not change while Il6 expression increased. It can be concluded that a combination of two long-lasting adverse factors, O. felineus infection and repeated social defeat stress, worsens not only the hepatic but also brain state, as evidenced behaviorally by disturbances of the startle response in mice.

Effects of supramolecular complexation of praziquantel with disodium glycyrrhizinate on the liver fluke Opisthorchis felineus: An in vitro and in vivo study.

Millions of people worldwide have a chronic infection with the liver fluke Opisthorchis felineus, which causes opisthorchiasis in humans and animals. The only known effective drug for this disease is praziquantel (PrzQ); however, its efficacy is below 100%, and it has some adverse effects. In this study, a water-soluble complex of PrzQ with a disodium salt of glycyrrhizic acid (disodium glycyrrhizinate; Na2GA) in a 1:10 ratio (PrzQ:GA) allowed the PrzQ dose to be decreased 11-fold for effective killing of O. felineus. An in vitro experiment showed a sufficient anthelmintic efficiency of PrzQ:GA against both adult and juvenile O. felineus individuals. A Syrian golden hamster model of opisthorchiasis revealed a considerable anthelmintic effect at all tested PrzQ:GA doses (50, 100, 200, 400, and 1100 mg/kg) with the best performance at 400 and 1100 mg/kg. Further comparison of the effects of PrzQ (400 mg/kg) and PrzQ:GA (400 mg/kg) regarding the state of the host indicated significant advantages of the latter. Histological examination showed that PrzQ:GA was better at decreasing the O. felineus-induced inflammatory infiltration (as compared with PrzQ alone) as well as interfered with the development of epithelial dysplasia and metaplasia in large bile ducts and cholangiofibrosis. Both PrzQ and PrzQ:GA decreased the number of myeloid (CFU-GM) and erythroid (BFU-E + CFU-E) colonies induced by O. felineus infection. The drugs had no negative effect on the animal behavior in an open field test 2-4 h after administration. Thus, PrzQ:GA proved to be a good anthelmintic agent having no evident adverse effects on the host, thereby suggesting that further preclinical and clinical trials would be warranted.

Social defeat stress exacerbates the blood abnormalities in Opisthorchis felineus-infected mice.

A model of chronic opisthorchiasis combined with social stress is examined; this situation is more likely for humans and animals than a separate impact of the infectious factor. For this purpose, we evaluated the effects of Opisthorchis felineus ("OP" group) and 30-day social stress (confrontations between males, "SS" group) alone and in combination ("OP + SS" group) in inbred C57BL/6 male mice and compared these effects according to the parameters listed below. The animals exposed to neither factor formed the control group ("CON"). All animals were assayed for blood biochemical parameters, changes in blood cell composition, and pattern of bone marrow hematopoiesis. By the end of the experiment, we have observed crucial effects of the two factors on the blood and liver of "OP" and "OP + SS". Eosinophil and basophil counts increased and relative segmented neutrophil and monocyte counts decreased in "OP + SS" mice on the background of activated myelopoiesis, mainly determined by social stress. Despite depressed erythropoiesis, "OP" mice displayed no changes in the relative peripheral erythrocyte counts. On the contrary, social stress, which stimulated erythropoiesis in "SS" and "OP + SS" mice, was accompanied by a decrease in the relative erythrocyte counts and hematocrit. Hepatosplenomegaly was observed on the background of these two impacts. Changes in transaminase (ALT and AST) and alkaline phosphatase activities as well as an increase in cholesterol and product of lipid peroxidation suggest a pronounced destruction of the liver. Altogether, social stress exacerbates many of the assayed blood parameters in the mice infected with the liver fluke.

Molecular Adaptations to Social Defeat Stress and Induced Depression in Mice.

Chronic stress is a risk factor for major depression. Social defeat stress is a well-validated murine model of depression. However, little is known about the gene activity dynamics during the development of a depression-like state. We analyzed the effects of social defeat stress of varying duration (10 and 30 days) on the behavioral patterns and prefrontal-cortex transcriptome of C57BL/6 mice. The 10-day exposure to social defeat stress resulted in a high level of social avoidance with no signs of depression-associated behavior. Most animals exposed to 30 days of social defeat stress demonstrated clear hallmarks of depression, including a higher level of social avoidance, increased immobility in the forced swimming test, and anhedonic behavior. The monitoring of transcriptome changes revealed widespread alterations in gene expression on the 10th day. Surprisingly, the expression of only a few genes were affected by the 30th day of stress, apparently due to a reversal of the majority of the early stress-induced changes to the original basal state. Moreover, we have found that glucocorticoid-sensitive genes are clearly stimulated targets on the 10th day of stress, but these genes stop responding to the elevated corticosterone level by the 30th day of stress. The majority of genes altered by the 30-day stress were downregulated, with the most relevant ones participating in chromatin modifications and neuroplasticity (e.g., guanine nucleotide exchange factors of the Rho-family of GTPases). Very different molecular responses occur during short-term and long-term social stress in mice. The early-stress response is associated with social avoidance and with upregulation and downregulation of many genes, including those related to signal transduction and cell adhesion pathways. Downregulation of a few genes, in particular, genes for histone-modifying methyltransferases, is a signature response to prolonged stress that induces symptoms of depression. Altogether, our data show that the development of depression under social stress conditions is correlated with suppression of the overactive molecular response to induced stress, involving gene regulatory resistance to glucocorticoid molecules, potentially via a chromatin remodeling mechanism.

Combined effects of social stress and liver fluke infection in a mouse model.

The effects of two influences, social stress and acute opisthorchiasis, were investigated in inbred C57BL/6J male mice. In the model of social stress, mice were repeatedly attacked and defeated by aggressive outbred ICR male mice and were in continuous sensory contact with an aggressive conspecific mouse in their home cage for 20 days. Acute opisthorchiasis was provoked by invasion of Opisthorchis felineus (50 larvae per animal) on the fourth day after the social stress was induced. Simultaneous action of both factors caused the hypertrophy of adrenal glands, as well as elevated the activity of cathepsins B and L in the spleen. This effect on the activity of the cysteine proteases in the hippocampus and hypothalamus following O. felineus invasion was the predominant result of simultaneous action with social stress. Acute opisthorchiasis, social stress, and their combination caused an increase in the level of blood IL-6 in approximately 30% of the animals. Social stress induced a more pronounced effect on mouse plus-maze behavior than O. felineus invasion. Our results suggest a more severe negative effect of the simultaneous influence of both factors on most of the parameters that were investigated.

Anxiety and ethanol consumption in victorious and defeated mice; effect of kappa-opioid receptor activation.

Alcohol consumption and addiction have been related to anxiety and the anxiolytic effect of ethanol. It has been shown in mice that losers with repeated experience of social defeats are more anxious than winners with repeated experience of victories. Mice with a different social status were tested for their oral ethanol consumption using a free two bottle choice paradigm and for their social approach behaviour after ethanol consumption using the partition test, in which anxiety is an important component. In addition, the sensitivity of the animals for the kappa-opioid receptor agonist U-50,488H (2.5 mg/kg, s.c.) was assessed using the partition test, in which this drug has been shown to induce anxiolytic-like effects. Further, the effect of daily treatment with U-50,488H for 8 days on ethanol consumption was tested in animals that had consumed ethanol and were subjected during these 8 days to a period of 5 days of interruption of ethanol supply and subsequently to a period of 3 days of renewed access to ethanol. Losers consumed more ethanol than winners. Consumption of ethanol was accompanied by a decrease of anxiety level, as evidenced by an increased approach behaviour in the partition test. U-50,488H stimulated ethanol consumption after a period of 5 days of interruption of ethanol supply and drug treatment in the losers, but not in the winners. U-50,488H increased approach behaviour in the losers not consuming ethanol and decreased this behaviour in the winners, especially in those that had consumed ethanol. It is postulated that U-50,488H acts as a partial agonist in this respect. The increased anxiety may be related to the enhanced ethanol consumption in the losers, which may be of relevance for the etiology of alcohol addiction.

Modulation of anxiety-related behaviors by mu- and kappa-opioid receptor agonists depends on the social status of mice.

This study was aimed to determine the effects of mu- and kappa-opioid receptor activation in relation to the social status of mice, being a winner with repeated experience of victories or a loser with repeated experience of social defeats. The behaviors of the animals were assessed in a social encounter test measuring the communicative behavior towards a familiar and an unfamiliar partner behind a perforated transparent partition (partition test) and in an elevated plus-maze test estimating the anxiety level of mice. Placebo and graded doses of the mu-opioid receptor agonist DAMGO (0.5 and 2 mg/kg s.c.) and the kappa-opioid receptor agonist U-50,488H (0.6, 1.25, and 2.5 mg/kg s.c.) were administered to the control mice, winners and losers in two experiments. In the partition test, the winners spent somewhat more time and the losers less time than the controls in the vicinity of their partner probably related to a lower and higher level of anxiety respectively. In the plus-maze test the losers appeared to have a somewhat higher anxiety level than the controls and winners. In both tests DAMGO produced anxiogenic-like effects in the winners and the controls, but not in the losers. Winners hardly responded to treatment with U-50,488H, while the losers responded dose dependently with an anxiolytic-like effect in both tests. It is concluded that anxiety-like responses in mice are differentially affected by stimulation of mu- and kappa-opioid receptors and that the effects depend on the social status of the animals.

Effects of chronic treatment with ipsapirone and buspirone on the C57BL/6J strain mice under social stress.

We experimented on inbred C57BL/6J strain mice who experienced social stress caused by defeat in inter-male confrontations for 20 days. From the fifth fight on, some mice were injected with ipsapirone (3 mg/kg), and some with buspirone (1 mg/kg) on a daily basis, for 14 days. Post-treatment behavior was examined in the plus-maze, partition, and Porsolt forced swim test (Porsolt's test). Each of these drugs had anxiolytic effects in the plus-maze, suggesting that they reduce state anxiety. Neither had any effect in the partition test, which provides further support to the hypothesis that normally the C57BL/6J strain mice have a high level of trait anxiety and for that reason they did not respond to the drugs. Chronic treatment with neither drug had any effect in the Porsolt's test. It is proposed that ipsapirone and buspirone fail to alleviate the depressive-like behaviors in the C57BL/6J mice because of a high level of trait anxiety, which might be inherent to this mouse strain.

Association between experience of aggression and anxiety in male mice.

The sensory contact technique increases aggressiveness in male mice and allows an aggressive type of behavior to be formed as a result of repeated experience of social victories in daily agonistic confrontations. In the low aggressive and high emotional mice of CBA/Lac strain, repeated positive fighting experience leads to increased plus maze anxiety in the winners after 10 days of experience of victories and much more after 20 days. Behavioral reactivity to other conspecifics was significantly increased as revealed by the parameters of partition test, which measures aggressive motivation in the winners. Thus, anxiety as a consequence of repeated experience of aggression is associated with the increase of aggressive motivation in CBA/Lac mice. It is concluded, that: (1) Repeated experience of aggression provokes the development of anxiety in male mice. (2) The level of anxiety as well as its behavioral realization depends on the duration of aggressive experience and genetic strain. Genetically defined features of innate anxiety (trait or state) in individuals may determine the kind of association between aggressive experience, aggressive motivation and anxiety.