Protective effect of Thunbergia laurifolia extract on hemolysis during Plasmodium berghei infection

This study was aimed to investigate the efficacy of Thunbergia laurifolia leaf extract to protect hemolysis in mice infected with Plasmodium berghei. Aqueous leaf extract of T. laurifolia was freshly prepared, and total polyphenol was then measured using Folin-Ciocalteu reagent method. For in vivo test, ICR mice were given intraperitoneally with this extract (1,000 mg/kg) once a day for four consecutive days and subsequently inoculated with 1?×?10⁶ parasitized erythrocytes of P. berghei ANKA by intraperitoneal injection for 8 days. The results showed that hemolysis was inhibited as indicated by %hematocrit (%Hct) which was normal in infected mice treated with T. laurifolia extract. Untreated and pyrimethamine-treated controls showed decreasing %Hct. Moreover, no any toxic signs were observed in normal mice treated with this extract. We conclude that T. laurifolia leaf extract clearly protects hemolysis during P. berghei infection in mice.     

Antimalarial, hematological, and antioxidant effects of methanolic extract of Terminalia avicennioides in Plasmodium berghei-infected mice

Terminalia avicennioides Guill. & Perr. (Combretaceae) is used traditionally to treat malaria in Nigeria. To establish its efficacy, methanolic extract of T. avicennioides bark was investigated for antimalarial activity against Plasmodium berghei (NK-65) in mice. Twenty-five mice in five groups were used for this study. Group 1 was uninfected normal control. Twenty mice infected with P. berghei were grouped as untreated negative control (group 2), 5 mg/kg b.w. p.o. artesunate-treated positive control (group 3), and 100 and 200 mg/kg b.w. p.o. T. avicennioides-treated infected mice (groups 4 and 5, respectively). Four-day suppressive effects on P. berghei and hematological and oxidative statuses of the mice were assessed. Suppression of parasitemia by artesunate and methanolic extract of T. avicennioides (at 100 and 200 mg/kg b.w.) after 1 day of treatment was 10, 18, and 11 % respectively; at day 5, the level of suppression was 77, 82, and 84 % respectively. P. berghei infection decreased hemoglobin, red blood cell, and lymphocyte counts and increased neutrophil count; artesunate and medicinal plant treatment restored these parameters to normal control levels. Also, artesunate and medicinal plant treatment of infected mice significantly (p?<?0.05) increased serum and liver superoxide dismutase activities and significantly (p?<?0.05) reduced serum malondialdehyde concentration compared to untreated infected mice. The antimalarial effect of T. avicennioides is comparable to that of artesunate. The restoration of oxidative and hematological statuses, to normal values by T. avicennioides, may provide better protection against the malaria severity and complications.     

In vivo antimalarial activity of Trichilia megalantha harms extracts and fractions in animal models

The crude methanol extracts of leaf, stem bark, root bark and stem bark fractions of Trichilia megalantha (Meliaceae) were screened for in vivo antimalarial activities in mice against a chloroquine resistant Plasmodium berghei berghei ANKA clone using the 4-day suppressive test procedure. Chloroquine diphosphate was used as the positive control. The extracts demonstrated intrinsic antimalarial property. Of all the seven extracts studied, the stem bark gave the highest activity. At 200 mg/kg of mouse, the stem bark extract had complete suppression of parasite growth (100 %). Least activity was observed for the leaf extract, while the root bark had a parasite suppression of 98.4 % at 800 mg/kg comparable to that of Chloroquine. Percentage suppression of parasite growth on day 4 post-infection ranged from 3.1 to 96.1 % in mice infected with P. berghei and treated with extracts and fractions of T. megalantha when compared with chloroquine diphosphate, the standard reference drug which had a chemosuppression of 96.2 %. At 400 mg/kg, the stem bark chloroform fraction was the most active fraction with 89.1 % parasite growth suppression followed by the ethyl acetate fraction (76.4 %), hexane soluble fraction (54.8 %) and methanol fraction (20.5 %). The mean survival time of mice that received extract ranged from 8.75?±?0.65 to 26.0?±?1.2 days (increased as the dose increases to 800 mg/kg), which was statistically significant, except the lowest dose (100 mg/kg) compared to the negative control group mice (9.45?±?0.6 days). The animals that were treated with Chloroquine had mean survival time of 23.5?±?1.2 days     

Bioassay-guided isolation and characterization of active antiplasmodial compounds from Murraya koenigii extracts against Plasmodium falciparum and Plasmodium berghei

Malaria is an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armoury of drugs for the treatment of malaria. This underscores the continuing need for the discovery and development of new effective and safe antimalarial drugs. To evaluate the in vitro and in vivo antimalarial activity of the leaf ethyl acetate extract of Murraya koenigii, bioassay-guided chromatographic fractionation was employed for the isolation and purification of antimalarial compounds. The in vitro antimalarial activity was assayed by the erythrocytic stages of chloroquine-sensitive strain of Plasmodium falciparum (3D7) in culture using the fluorescence-based SYBR Green I assay. The in vivo assay was done by administering mice infected with Plasmodium berghei (NK65) four consecutive daily doses of the extracts through oral route following Peter’s 4-day curative standard test. The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in untreated control with those of treated mice. Cytotoxicity was determined against HeLa cells using MTT assay. Histopathology was studied in kidney, liver and spleen of isolated compound-treated Swiss albino mice. The leaf crude ethyl acetate extract of M. koenigii showed good in vitro antiplasmodial activity against P. falciparum. The in vivo test of the leaf crude ethyl acetate extract (600 mg/kg) showed reduced malaria parasitaemia by 86.6 % against P. berghei in mice. Bioassay-guided fractionation of the leaf ethyl acetate extract of M. koenigii led to the isolation of two purified fractions C3B2 (2.84 g) and C3B4 (1.97 g). The purified fractions C3B2 and C3B4 were found to be active with IC₅₀ values of 10.5?±?0.8 and 8.25?±?0.2 μg/mL against P. falciparum, and in vivo activity significantly reduced parasitaemia by 82.6 and 88.2 % at 100 mg/kg/body weight on day 4 against P. berghei, respectively. The isolated fractions C3B2 and C3B4 were monitored by thin-layer chromatography until a single spot was obtained with R _f values of 0.36 and 0.52, respectively. The pure compounds obtained in the present investigation were subjected to UV–visible spectroscopy, Fourier transformer infrared spectroscopy, 1D and 2D ¹H-Nuclear magnetic resonance (NMR), ¹³C NMR, DEPT, COSY and Mass spectral analysis. Based on the spectral analysis, it is concluded that the isolated compounds were myristic acid (C3B2) and β-caryophyllene (C3B4). The cytotoxic effect of myristic acid and β-caryophyllene showed the TC₅₀ values of >100 and 80.5 μg/mL, respectively against HeLa cell line. The histopathology study showed that protection against nephrotoxicity of kidney, hepatic damage of liver and splenocytes protection in spleen was achieved with the highest dose tested at 100 mg/kg/body weight. The present study provides evidence of antiplasmodial compounds from M. koenigii and is reported for the first time.     

Prothrombotic State in Asthma Is Related to Increased Levels of Inflammatory Cytokines,IL-6 and TNFα, in Peripheral Blood

Recently, we have reported that asthma is associated with enhanced plasma thrombin formation and impaired fibrinolysis. The mechanisms underlying the prothrombotic state in this disease are unknown. Our aim was to investigate whether prothrombotic alterations in asthmatics are associated with inflammation. We studied 164 adult, white, stable asthmatics and 72 controls matched for age, sex, body mass index (BMI), and smoking. Plasma tumor necrosis factor α (TNFα), interleukin (IL)-6, and serum periostin were evaluated using ELISAs, and their associations with thrombin generation, fibrinolytic capacity, expressed as clot lysis time (CLT), and platelet markers were later analyzed. Asthma was characterized by 62% higher plasma IL-6 and 35% higher TNFα (both, p < 0.0001). Inflammatory cytokines were higher in sporadic and persistent asthmatics compared to controls, also after adjustment for potential confounders. IL-6 was inversely related to the forced expiratory volume in 1 s/vital capacity (FEV₁/VC) spirometry index after correction for age, sex, and BMI. IL-6 and TNFα were associated with C-reactive protein in asthmatics (β = 0.6 [95% CI, 0.54–0.67] and β = 0.33 [95% CI, 0.25–0.41], respectively) and controls (β = 0.43 [95% CI, 0.29–0.57] and β = 0.33 [95% CI, 0.18–0.48], respectively). In asthma, IL-6 and TNFα positively correlated with the endogenous thrombin potential (β = 0.35 [95% CI, 0.28–0.42] and β = 0.15 [95% CI, 0.07–0.23], respectively) but not with CLT or platelet markers. However, TNFα predicted CLT in a multiple linear regression model. Periostin was not associated with any hemostatic parameters. Enhanced thrombin generation is driven in asthma by a systemic inflammatory state mediated by IL-6 and to a lesser extent TNFα, however, not periostin. TNFα might contribute to impaired fibrinolysis.     

Antimalarial efficacy,cytotoxicity, and genotoxicity of methanolic stem bark extract from Hintonia latiflora in a Plasmodium yoelii yoelii lethal murine malaria model

Traditional medicines have been used to treat malaria for thousands of years and are the source of artemisinin and quinine derivatives. With the increasing levels of drug resistance, the high cost of artemisisnin-based combination therapies, and fake antimalarials drugs, traditional medicine have become an important and sustainable source of malaria treatment. For the benefit of those who use traditional medicine to treat malaria, there is an urgent need to study the efficacy and toxicity of herbal remedies. Hintonia latiflora stem bark infusions are use in Mexican traditional medicine to treat malaria, diabetes, and gastrointestinal diseases. Its efficacy in the treatment of complicated malaria and its ability to generate DNA damage to the host is not fully evaluated. In our search for antimalarial natural products, in the present study, we tested the efficacy of H. latiflora stem bark methanolic extract (HlMeOHe) in CD1 male mice infected with lethal Plasmodium yoelii yoelii and its in vivo cytotoxicity and genotoxicity. To assess the antimalarial activity, the extract was evaluated in a 4-day test scheme in oral doses of 1,200, 600, and 300 mg/kg prior acute toxicity test; oral chloroquine (15 mg/kg) was used as positive control. The ability of 1,200 mg/kg of HlMeOHe to induce cytotoxicity and DNA damage in the peripheral blood of mice was assessed using a fluorochrome-mediated viability test and the micronucleus (MN) assay; N-ethyl-N-nitrosourea (ENU) was used as a positive control. HlMeOHe median acute toxicity (LD₅₀) was 2,783.71 mg/kg and LD₁₀ was 1,293.76 mg/kg (taken as the highest work dose). Plasmodium yoelii yoelii-infected mice in the untreated control group died between 6 and 7 days post-infection (PI) with parasitemia over 70 %. Even though mice treated with 600 and 300 mg/kg showed a chemosuppression percentage of total parasitemia of 99.23 and 23.66, respectively, animals in both groups died 6 to 7 days PI with parasitemia over 45 %. A 4-day dosage of 1,200 mg/kg of the extract showed, in the P. yoelii yoelii-infected mice, a 100 % chemosuppression of total parasitemia on 5 days PI and a 23 days survival time with a mean parasitemia of 23.6 % at the date of death. Only mice treated with chloroquine survived until the end of the experiment. Cell viability was not affected. The average number of micronuclei in the treated mice increased significantly (P?<?0.05) to 4.8 MN when compared with the untreated control group (0.9 MN). The results obtained in this study showed that the infection outcome of P. yoelii yoelii-infected mice is affected by HlMeOHe. Although a concentration of 1,200 mg/kg of HlMeOHe is suitable to use in the treatment of malaria fever, slowed down the parasite replication, retarded the patency time, and increased the infected P. yoelii yoelii mice survival time, its chemical composition should be studied in detail in order to reduce its genotoxic potential.     

Modulation of alveolar macrophage innate response in proinflammatory-, pro-oxidant-, and infection- models by mint extract and chemical constituents: Role of MAPKs

There is a continuing need for discovering novel primary or adjunct therapeutic agents to treat inflammatory conditions and infections. Natural products have inspired the discovery of several modern therapeutics; however, there is a paucity of mechanistic information on their mode of action. This study investigated the therapeutic potential and mode of action of corn mint’s (Mentha arvensis) leaf extract (ME) in alveolar macrophages (AMs) challenged with model pro-inflammatory (LPS), pro- oxidant (LPS or H₂O₂), and infection (Mycobacterium) agents and contribution of its dominant constituents rosmarinic acid, l-menthol, and l-menthone. LPS-induced inflammatory response in the murine AM cell line MH-S was significantly reduced in terms of pro-inflammatory cytokines (TNF-α, IL-1α) and nitric oxide (NO) when pre- or post-treated with ME. The ME pretreatment of macrophages led to a significant increase ( P≤ 0.05) in phagocytic activity toward Mycobacterium smegmatis and a greater pathogen clearance in 24 h in both ME pre-treated (P ≤ 0.05) and post-treated cells. Significant attenuation (P ≤ 0.01) of reactive oxygen species (ROS) production in LPS- or H₂O_2-treated macrophages by pretreatment with whole mint extract (ME) was accounted for in part by the mint constituents rosmarinic acid and l-menthone. Attenuation of pro-inflammatory response by ME pretreatment coincided with the significant reduction in total and phosphorylated JNK1/2, decrease in total p38, and increase in phospho-ERK1/2 thereby implying a role of differential modulation of MAPKs. Taken together, the results demonstrate that corn mint leaf components cause potent anti-inflammatory, anti-oxidant, and anti–infection effects in AMs via suppression of the production of cytokines/soluble mediators and ROS and increased pathogen clearance, respectively. To our knowledge, this is the first report on the mode of action of corn mint targeting the alveolar macrophages and on the potential role of MAPKs in immunomodulation by this product.     

Macrophage Migration Inhibitory Factor contributes to drive phenotypic and functional macrophages activation in response to Toxoplasma gondii infection

Cytokines are small molecules secreted by numerous cells. Macrophage Migration Inhibitory Factor (MIF) is a cytokine initially described due to its function of inhibiting random macrophage migration. Currently, new functions have been described for MIF, such as stimulating inflammatory functions in response to infections by microorganisms including, Toxoplasma gondii. However, the primordial MIF function related to macrophages has been little addressed. The main purpose of the study was to recapitulate MIF function on macrophages in response to T. gondii infection. To achieve this goal, peritoneal macrophages were collected from C57BL/6WT and Mif1^-/- mice after recruitment with thioglycolate. Macrophages were cultured, treated with 4-Iodo-6-phenylpyrimidine (4-IPP), and infected or not by T. gondii for 24 h. Following this, the culture supernatant was collected for cytokine, urea and nitrite analysis. In addition, macrophages were evaluated for phagocytic activity and T. gondii proliferation rates. Results demonstrated that T. gondii infection triggered an increase in MIF production in the WT group as well as an increase in the secretion of IL-10, TNF, IFN-γ, IL-6 and IL-17 in the WT and Mif1^-/- macrophages. Regarding the comparison between groups, it was detected that Mif1^-/- macrophages secreted more IL-10 compared to WT. On the other hand, the WT macrophages produced greater amounts of TNF, IFN-γ, IL-6 and IL-17. Urea production was more pronounced in Mif1^-/- macrophages while nitrite production was higher in WT macrophages. T. gondii showed a greater ability to proliferate in Mif1^-/- macrophages and these cells also presented enhanced phagocytic activity. In conclusion, T. gondii infection induces macrophage activation inciting cytokine production. In presence of MIF, T. gondii infected macrophages produce pro-inflammatory cytokines compatible with the M1 activation profile. MIF absence caused a dramatic reduction in pro-inflammatory cytokines that are balanced by increased levels of urea and anti-inflammatory cytokines. These macrophages presented increased phagocytic capacity and shared features activation with the M2 profile.     

Natural polyamine inhibits mouse skin inflammation and macrophage activation

Objective

Natural polyamines are some of the most abundant polycationic molecules in eukaryotic cells, regulating gene expression. Polyamines have been reported to possess anti-inflammatory activities in many model inflammation systems. However, there is no report on their role in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced dermal edema.

Methods

Mouse ear edema was induced by TPA. Edema biopsies were investigated using H&E staining. Levels of nitric oxide (NO) were determined using the Griess reaction. Tumor necrosis factor α (TNFα) and interleukin (IL)-1β levels in cell supernatants were measured by TNFα and IL-1β ELISA kits.

Results

Spermidine and spermine caused significant decreases in ear thickness, water content, and neutrophil infiltrations in comparison with negative control (p < 0.05). External polyamines reduced the levels of inflammatory mediators such as NO, TNFα, and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. Spermine had a higher inhibitory effect on the production of cytokines such as IL-1β and TNFα in LPS-stimulated murine macrophages compared to other polyamines.

Conclusion

Our findings clearly demonstrated that polyamines are involved in the anti-inflammatory effect by reducing dermal edema thickness and other inflammatory mediators like NO and cytokines in a dose-dependent manner.     

Role of the Aryl Hydrocarbon Receptor in the Immune Response Profile and Development of Pathology during Plasmodium berghei Anka Infection

Infection with Plasmodium falciparum may result in severe disease affecting various organs, including liver, spleen, and brain, resulting in high morbidity and mortality. Plasmodium berghei Anka infection of mice recapitulates many features of severe human malaria. The aryl hydrocarbon receptor (AhR) is an intracellular receptor activated by ligands important in the modulation of the inflammatory response. We found that AhR-knockout (KO) mice infected with P. berghei Anka displayed increased parasitemia, earlier mortality, enhanced leukocyte-endothelial cell interactions in the brain microvasculature, and increased inflammation in brain (interleukin-17 [IL-17] and IL-6) and liver (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) compared to infected wild-type (WT) mice. Infected AhR-KO mice also displayed a reduction in cytokines required for host resistance, including TNF-α, IL-1β, and IFN-γ, in the brain and spleen. Infection of AhR-KO mice resulted in an increase in T regulatory cells and transforming growth factor β, IL-6, and IL-17 in the brain. AhR modulated the basal expression of SOCS3 in spleen and brain, and P. berghei Anka infection resulted in enhanced expression of SOCS3 in brain, which was absent in infected AhR-KO mice. These data suggest that AhR-mediated control of SOCS3 expression is probably involved in the phenotype seen in infected AhR-KO mice. This is, to our knowledge, the first demonstration of a role for AhR in the pathogenesis of malaria.     

Effects of dexmedetomidine on early and late cytokines during polymicrobial sepsis in mice

Objective

We investigated whether dexmedetomidine provided protective effects on cecal ligation and puncture (CLP)–induced septic mice, through suppressing the expression of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6)] and high mobility group box 1 (HMGB1).

Methods

The model of sepsis was set up by CLP in 136 male BALB/c mice (40 mice for survival studies and 96 for cytokine studies) which were divided into four groups, including a C, CLP, DEX + CLP and CLP + DEX group. The serum levels of TNF-α, IL-6 and HMGB1 were detected at 6, 12, 24 and 48 h after operations, and lung HMGB1 mRNA were analyzed at 24 and 48 h. The mortality rates were calculated 7 days after the operations.

Results

The mortality rates 7 days after operations were significantly lower in the CLP + DEX (50 %) and DEX + CLP (30 %) groups than in the CLP group (90 %). Serum concentrations of IL-6 and TNF-α decreased significantly in dexmedetomidine administration groups compared with the CLP group. The levels of HMGB1 and lung HMGB1 mRNA were lower in the dexmedetomidine administration groups than in the CLP group. There was a significant correlation between lung HMGB1 mRNA and serum HMGB1(r = 0.858).

Conclusions

Dexmedetomidine could reduce the mortality rate and inhibit pro-inflammatory cytokine responses during polymicrobial sepsis in mice.     

Is there a reduced sensitivity of dihydroartemisinin against praziquantel-resistant Schistosoma japonicum?

Praziquantel is currently the only drug of choice for the treatment of human schistosomiases. However, it has been proved that Schistosoma japonicum subjected to drug pressure may develop resistance to praziquantel. To evaluate the efficacy of dihydroartemisinin against praziquantel-resistant S. japonicum, mice infected with a praziquantel-resistant isolate and a praziquantel-susceptible isolate of S. japonicum were treated with dihydroartemisinin at a single oral dose of 300 mg/kg given once on each of 35–36 post-infection days, while infected but untreated mice served as controls. All mice were sacrificed 50 days post-infection, and the worm burden reductions were estimated. Administration of dihydroartemisinin at a single oral dose of 300 mg/kg on each of 35–36 post-infection days reduced total worm burdens of 69.8 % and female worm burdens of 86 % in mice infected with the praziquantel-susceptible isolate, and total worm burdens of 66.1 % and female worm burdens of 85.1 % in mice infected with the praziquantel-resistant isolate (both P values?>?0.05). It is concluded that the sensitivity of artemisinin derivative dihydroartemisinin does not reduce in praziquantel-resistant S. japonicum.     

Effect of root bark extract of Berberis vulgaris L. on Leishmania major on BALB/c mice

Leishmaniasis is one of the most important diseases transmitted by arthropod. This disease is present in 88 countries. Approximately 400 million people are at risk, and 12 million are involved. We aimed to examine the application of ethanolic extract of the root bark of Berberis vulgaris L. for treatment of mice infected with cutaneous leishmaniasis. At first, 40 BALB/c mice were infected to Leishmania major promastigotes and were divided in two groups A and B. Then, each of A and B groups were divided to two subgroups. Mice from subgroup A1 were treated with 10 % root bark alcoholic extract, and mice from subgroup A2 were treated with only alcohol (control). Mice from subgroup B1 were treated with 20 % root bark alcoholic extract, and mice from subgroup B2 were treated with only alcohol (control). The 90 % recovery was found in the mice treated with 20 % root bark extract, and 55 % recovery was found with 10 % root bark extract, but in the control group, 0 % recovery was found. The results of our study showed that the lotion of root bark extract has good suppression effects on parasites. Therefore, it might be a pro for developing new antileishmanial drugs.     

Swertiamarin ameliorates inflammation and osteoclastogenesis intermediates in IL-1β induced rat fibroblast-like synoviocytes

Objective and design

Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1β induced fibroblast-like synoviocytes (FLS).

Methods

The FLS were isolated from Freund’s Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1β. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10–50 μg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h).

Results

IL-1β induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03 %) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22 %) and NO production (82.06 ± 3.91 % at 50 μg/ml) in a dose-dependent manner. It also significantly (P < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly (P < 0.05) attenuated the release of the same in time dependent mode.

Conclusion

These findings suggest that treatment with swertiamarin attenuated IL-1β induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.     

Pathogenicity ofSerpulina hyodysenteriae:in vivoinduction of tumor necrosis factor and interleukin-6 by a serpulinal butanol/water extract (endotoxin)

Lipopolysaccharide (LPS) of Gram-negative bacteria is a classic inducer of inflammatory cytokines. In the present experiments, LPS-like (phenol/water extract) or endotoxin-like (butanol/water extract) preparations fromSerpulina hyodysenteriaewere examined for their ability to induce serum tumor necrosis factor (TNF) or interleukin (IL)-6 bioactivity in mice and swine.Serpulina hyodysenteriaeendotoxin (butanol/water extract) elicited increased serum TNF activity in mice, although serpulinal endotoxin was at least 10 times less potent than the LPS preparations ofE. coliorS. typhimuriumon a weight basis for induction of TNF bioactivity.S. hyodysenteriaeLPS induced lower levels of serum TNF in mice thanS. hyodysenteriaeendotoxin. In contrast, pigs injected withS. hyodysenteriaeendotoxin demonstrated no increase in serum TNF activity. However, an induction of IL-6 bioactivity was observed in serum samples from pigs injected with serpulinal endotoxin. In pigs, the serpulinal preparations were five times less potent (on a weight basis) thanE. coliorS. typhimuriumLPS for induction of IL-6 bioactivity. These data indicate that serpulinal endotoxin, although less bioactive thanE. coliorS. typhimuriumLPS, is nonetheless capable of inducing thein vivoproduction of specific pro-inflammatory cytokines.     

Viper venom-induced oxidative stress and activation of inflammatory cytokines: a therapeutic approach for overlooked issues of snakebite management

Background and objective

The snakebite mortality rate has been significantly reduced due to effective anti-venin therapy. The intravenously infused anti-venom will neutralize free and target-bound toxins but fails to neutralize venom-induced inflammation and oxidative stress, as the antigen–antibody complex itself is pro-inflammatory. Therefore, an auxiliary therapy is necessary to treat secondary/overlooked envenomation complications.

Materials and methods

Blood samples from healthy donors were treated with viper venom (100 μg/ml) for 2 h. The venom-induced inflammation, oxidative damage and effect of crocin pre-treatment were determined by assessing the serum levels of cytoplasmic, lysosomal and oxidative stress markers along with pro-inflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and cyclo-oxygenase (COX)-2.

Results

Significantly increased stress markers, cytoplasmic, lysosomal and extracellular matrix-degrading enzymes as well as the pro-inflammatory mediators TNF-α, IL-1β, IL-6 and COX-2 indicated increased cellular damage but significantly reduced oxidative damage and inflammation in crocin pre-treated groups.

Conclusion

The data clearly suggest that venom-induced oxidative stress and inflammation is also responsible for oxidative burst and cell death in the circulation, which may worsen even after anti-venin therapy. Hence, the current study demands a supportive therapy in addition to anti-venin therapy to neutralize the overlooked issues of snakebite.     

Efficacy of artemether and artesunate in mice infected with praziquantel non-susceptible isolate of Schistosoma japonicum

Praziquantel is currently the only drug of choice for the treatment of human Schistosoma japonicum infections, and praziquantel-based chemotherapy has been proved to be generally effective to control the morbidity and reduce the prevalence and intensity of S. japonicum infections. However, the potential emergence of praziquantel resistance in S. japonicum seriously threatens the elimination of this neglected tropical disease in China. The purpose of this study was designed, in mouse animals, to evaluate the in vivo efficacy of artemether and artesunate against praziquantel non-susceptible S. japonicum. Mice infected with a praziquantel non-susceptible isolate and a praziquantel-susceptible isolate of S. japonicum were treated with artemether and artesunate at a single oral dose of 300 mg/kg given once on each of days 7–8 and 35–36 post-infection to assess the efficacy against juvenile and adult worms. Administration with artemether and artesunate at a single oral dose of 300 mg/kg on each of days 7–8 post-infection resulted in total worm burden reductions of 72.8 and 73.5 % in mice infected with praziquantel-susceptible S. japonicum, and 77.9 and 74.1 % in mice infected with the non-susceptible isolate (both P values >0.05), while the same treatments given on days 35–36 post-infection reduced total worm burdens by 71.4 and 69.6 % in mice infected with the susceptible isolate, and 75.3 and 69.6 % in mice infected with the non-susceptible parasite (both P values >0.05). It is concluded that there is no evidence for reduced susceptibility of artemether and artesunate in praziquantel non-susceptible S. japonicum.     

IM-133N modulates cytokine secretion by RAW264.7 and THP-1 cells

An indigenous herbal extract IM-133N containing extracts of Prosopis glandulosa Torr and Symplocos racemosa Roxb were evaluated for potential immunomodulatory effects using RAW264.7 and THP-1 cells. The incubation of the cells for 24?h with IM-133N over a dose range 0–125?µg/ml did not cause cytotoxicity that exceeded 10%. The results indicated that non-cytotoxic doses of IM-133N effectively up-regulated iNOS, TNFα, IL-6, IL-10, IL-8 and IFNγ gene expression in both the RAW264.7 and THP-1 cells. The results also indicated IM-133N elicited dose-related increases in nitric oxide (NO) and tumor necrosis factor (TNF)-α production by RAW264.7 or THP-1 cells. These results demonstrated that IM-133N could stimulate NO and induced pro-inflammatory cytokine expression by monocytes/macrophages. As clinical studies have shown IM-133N to be an effective immunomodulator without any adverse effects, the results of the present study provide further support for the potential use of this agent as an immunostimulant or as an immunotherapy adjuvant.     

Enhanced antimalarial effects of chloroquine by aqueous Vernonia amygdalina leaf extract in mice infected with chloroquine resistant and sensitive Plasmodium berghei strains

Background

The emergence and spread of Plasmodium falciparum with resistance to chloroquine (CQ), the safest and cheapest antimalarial drug coupled with the increasing cost of alternative drugs especially in developing countries have necessitated the need to optimize antimalarial actions of plant extracts and restore chloroquine efficacy.

Objective

The present study determines the ability of Vernonia amygdalina leaf extract to enhance the prophylactic and therapeutic efficacy of chloroquine against Plasmodium berghei malaria in mice.

Methods

Chloroquine sensitive (P. berghei^S) and resistant (P.berghei^R) ANKA clones of Plasmodium berghei maintained by serial passage in mice were used to develop respective experimental rodent malaria models based on intraperitoneal injection of 10⁶ parasitize erythrocyte suspension in PBS (pH 7.2) and subsequent development of parasitaemia. These models were then used to investigate the prophylactic enhancement of chloroquine (CQ) at 5 mg/kg via combination with selected doses (31.25, 62.5, 125mg/kbw) of Vernonia amygdalina leaf extracts using a 4-day suppression test. Effect of these combinations on the therapeutic efficacy of CQ at 30mg/kg over 3 days were evaluated. Treatment outcomes including parasite clearance (PCT) and rescrudescent time (RT) were compared with CQ-chlorpheniramine combination. The acute toxicity of the extract-CQ combinations was also determined enzymatically.

Results

Prophylatically, chloroquine (5mg/kg) in combination with vernonia extracts achieved a dose-dependent (57.2–72.7%) suppression of parasitaemia due to CQ sensitive and resistant P berghei strains in the experimental animals. Therapeutically, chloroquine (30mg/kg for 3 days) combined with vernonia to dose-dependently shorten the parasite clearance times (2.6–4.4 vs. 4.8 days; P < 0.05 for CQ-V62.5/125 combination), prolong the recrudescent times (8.9–18.9 vs. 7.2 days; P < 0.05) and improve day 14 cure rate (66.7–100 vs. 58.3%) in the treated P. berghei^S infected mice compared to CQ monotherapy. Whereas CQ monotherapy failed, resolution of parasitaemia due to the CQ resistant parasite with day 14 cure rates of 25 – 100% were also observed with these combinations. In therapeutic terms, the potencies of CQ-V125 combination were comparable to those of CQ-chlorpheniramine (0.25mg/kg, 12hourly, 7 days) in the infected animals. Toxicity testing indicates that these combinations elicited mild to - moderate increases in the liver enzymes measured when administered orally to mice for 7 days.

Conclusion

This study indicates that Vernonia amygdalina leaf extract dose - dependently restore the efficacy of CQ against CQ resistance P. berghei malaria in mice.