Erratum for “Effect of aqueous leaf extract of Thunbergia laurifolia on alcohol-induced liver injury in rats”

Purpose: To investigate the antioxidant and anti-inflammatory effects of aqueous leaf extract of T. laurifolia against alcoholic liver injury in rats. Methods: Male Wistar rats were administered either normal saline, ethanol (EtOH), normal saline with low or high dose T. laurifolia leaf extract (TL-LD or TL-HD), EtOH with TL-LD or EtOH with TL-HD. Blood biochemical indices: hepatic malondialdehyde (MDA) levels, liver histopathology, hepatic cytochrome P450 2E1 (CYP2E1), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and proinflammatory cytokines, including interleukin 1 beta (IL-1β) and tumor necrotic factor alpha (TNF-α) mRNA expressions, were determined using standard methods. Results: The leaf extract of T. Laurifolia decreased hepatic MDA levels, improved liver pathology and down-regulated mRNA expressions of CYP2E1, NADPH oxidase and pro-inflammatory cytokinesin ethanol-treated rats. Conclusion: These results demonstrate that aqueous extract of T. Laurifolia exerts hepatoprotective effect against alcoholic liver injury through a mechanism involving inhibition of oxidative stress and inflammation

Effect of aqueous leaf extract of Thunbergia laurifolia on alcohol-induced liver injury in rats

Purpose: To investigate the antioxidant and anti-inflammatory effects of aqueous leaf extract of T. laurifolia against alcoholic liver injury in rats. Methods: Male Wistar rats were administered either normal saline, ethanol (EtOH), normal saline with low or high dose T. laurifolia leaf extract (TL-LD or TL-HD), EtOH with TL-LD or EtOH with TL-HD. Blood biochemical indices: hepatic malondialdehyde (MDA) levels, liver histopathology, hepatic cytochrome P450 2E1 (CYP2E1), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and pro-inflammatory cytokines, including interleukin 1 beta (IL-1β) and tumor necrotic factor alpha (TNF-α) mRNA expressions, were determined using standard methods. Results: The leaf extract of T. Laurifolia decreased hepatic MDA levels, improved liver pathology and down-regulated mRNA expressions of CYP2E1, NADPH oxidase and pro-inflammatory cytokinesin ethanol-treated rats. Conclusion: These results demonstrate that aqueous extract of T. Laurifolia exerts hepatoprotective effect against alcoholic liver injury through a mechanism involving inhibition of oxidative stress and inflammation

Effect of a heme oxygenase-1 inducer on NADPH oxidase expression in alcohol-induced liver injury in male Wistar rats

Purpose: To investigated the effect of hemin, a heme oxygenase-1 (HO-1) inducer, on nicotinamide adenine dinucleotide phosphate oxidase (NOX) expression in rats with alcohol-induced liver injury.Methods: Male Wistar rats were randomly divided into four groups consisting of the control group, the ethanol (EtOH) group, the EtOH + zinc protoporphyrin IX (ZnPP-IX) group and EtOH + hemin group. Hepatic NOX gene expression and immunohistochemistry of hepatic NOX1 and NOX4 were investigated in week 4.Results: EtOH significantly increased levels of NOX. An immunohistochemical study demonstrated a high number of immunopositive hepatocytes for NOX1 in the EtOH group and EtOH + ZnPP-IX group compared with the control group. Hemin administration downregulated NOX gene expression and lowered the number of immunopositive hepatocytes for NOX1. In contrast, ZnPP-IX (HO-1 inhibitor) administration caused upregulation of NOX gene expression and increased the number of immunopositive hepatocytes for NOX1.Conclusion: HO-1 inducer, hemin, alleviates oxidative stress-induced alcoholic liver injury by reducing NOX, especially NOX1.Keywords: NADPH oxidase, Immunohistochemistry, Heme oxygenase-1, Hemin, Reactive oxygen species, Alcohol-induced liver diseas

Immunofluorescence study of cytoskeleton in endothelial cells induced with malaria sera

Background: Endothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines. Methods: Morphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ. Results: The four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of “spiking pattern” of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ. Conclusions: ECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity

Activation of nuclear factor kappa B in peripheral blood mononuclear cells from malaria patients

BACKGROUND: Malaria parasites and their products can activate a specific immune response by stimulating cytokine production in the host’s immune cells. Transcription nuclear factor kappa B (NF-κB) is an important regulator for the control of many pro-inflammatory genes, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). The activation and expression of NF-κB p65 in peripheral blood mononuclear cells (PBMCs) of malaria patients were investigated and correlated with the levels of IL-10 and TNF to study the nature of NF-κB p65 and its linkage to inflammatory cytokines. METHODS: The sample group comprised 33 patients admitted with malaria caused by Plasmodium vivax (n = 11), uncomplicated Plasmodium falciparum (n = 11), and complicated Plasmodium falciparum (n = 11). Peripheral blood was collected at admission and on day 7 for PBMC isolation. Healthy subjects were used as a control group. The expressions of NF-κB p65 in the PBMCs from malaria patients and the plasma levels of IL-10 and TNF were measured by using enzyme-linked immunosorbent assay (ELISA). The immunofluorescence technique was used to determine NF-κB nuclear translocation. RESULTS: At admission, patients with P. vivax and uncomplicated P. falciparum had significantly elevated phospho-NF-κB p65 levels in the PBMCs compared with those of healthy controls. However, patients with complicated P. falciparum malaria had decreased levels of phospho-NF-κB p65. On day 7 post-treatment, significantly increased phospho-NF-κB p65 was found in the PBMCs of patients with complicated P. falciparum, compared with healthy controls. The plasma level of IL-10 was elevated in day 0 in patients with complicated P. falciparum malaria and was found to be negatively correlated with phospho-NF-κB p65 level (r(s) = −0.630, p = 0.038). However, there was no correlation between phospho-NF-κB p65 expression and TNF level in patients with complicated P. falciparum malaria. CONCLUSIONS: This is the first report demonstrating alterations in NF-κB p65 activity in the PBMCs of malaria patients. The altered lower features of NF-κB p65 in the PBMCs of patients with complicated P. falciparum at admission could be due to a suppressive effect of high IL-10 associated with complicated P. falciparum malaria

Overexpression of Sphingosine Kinase-1 and Sphingosine-1-Phosphate Receptor-3 in Severe Plasmodium falciparum Malaria with Pulmonary Edema

Pulmonary edema (PE) is a major cause of pulmonary manifestations of severe Plasmodium falciparum malaria and is usually associated with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The sphingosine kinase-1 (SphK-1)/sphingosine-1-phosphate receptor-3 (S1PR-3) pathway has recently been reported to affect the pathogenesis of lung injury, but the expression of these proteins in the lungs of severe P. falciparum malaria patients has not been investigated. The cellular expression of SphK-1 and S1PR-3 in lung tissues from autopsied patients with P. falciparum malaria was investigated using immunohistochemistry (IHC). Lung tissues from patients who died of severe P. falciparum malaria were classified into two groups based on histopathological findings: those with PE (18 patients) and those without PE (non-PE, 19 patients). Ten samples of normal lung tissues were used as the control group. The protein expression levels of SphK-1 and S1PR-3 were significantly upregulated in endothelial cells (ECs), alveolar epithelial cells, and alveolar macrophages (AMs) in the lungs of severe P. falciparum malaria patients with PE compared to those in the non-PE and control groups (all p<0.001). In addition, the SphK-1 and S1PR-3 expression levels were significantly positively correlated in pulmonary ECs (rs=0.922, p<0.001), alveolar epithelial cells (rs=0.995, p<0.001), and AMs (rs=0.969, p<0.001). In conclusion, both the SphK-1 and S1PR-3 proteins were overexpressed in the lung tissues of severe P. falciparum malaria patients with PE, suggesting that SphK-1 and S1PR-3 mediate the pathogenesis of PE in severe malaria. Targeting the regulation of SphK-1 and/or S1PR-3 may be an approach to treat pulmonary complications in severe P. falciparum patients

Expression of sphingosine kinase 1 and sphingosine 1-phosphate receptor 3 in malaria-associated acute lung injury/acute respiratory distress syndrome in a mouse model.

This study aimed to investigate the expression of sphingosine kinase 1 (SphK-1) and sphingosine 1-phosphate receptor 3 (S1PR-3) in a mouse model of malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS). DBA/2 mice were infected with Plasmodium berghei ANKA to generate an experimental model of malaria-associated ALI/ARDS. The infected mice were divided into 2 groups based on the histopathological study of lung tissues: those with and those without ALI/ARDS. The expression of the SphK-1 and S1PR-3 proteins in the lung tissues was investigated using immunohistochemical staining and Western blot analysis. In addition, the S1P level was quantified in plasma and lung tissues using an enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the cellular expression of the SphK-1 and S1PR-3 proteins was significantly upregulated in endothelial cells, alveolar epithelial cells and alveolar macrophages in the lung tissues of malaria-infected mice with ALI/ARDS compared with those in the control groups. The increased expression of the SphK-1 and S1PR-3 proteins was confirmed using Western blot analysis. The concentration of S1P in plasma and lung tissues was significantly decreased in malaria-infected mice with ALI/ARDS compared with non-ALI/ARDS and control mice. Furthermore, increased expression of the SphK-1 and S1PR-3 proteins significantly correlated with lung injury scores and S1P concentrations in malaria-infected mice with ALI/ARDS. These findings highlight increased expression of SphK-1 and S1PR-3 in the lung tissues of malaria-infected mice with ALI/ARDS

Expression of 4-Hydroxynonenal (4-HNE) and Heme Oxygenase-1 (HO-1) in the Kidneys of Plasmodium berghei-Infected Mice

Acute kidney injury (AKI) is one of the most serious complications of severe Plasmodium falciparum malaria, but the exact pathogenic mechanisms of AKI in P. falciparum infection have not been clearly elucidated. We hypothesized that oxidative stress is a potential mediator of acute tubular necrosis in P. falciparum-infected kidneys. Therefore, this study aimed to investigate the histopathological changes and markers of oxidative stress in kidney tissues from mice with experimental malaria. DBA/2 mice were divided into two groups: the mice in the malaria-infected group (n = 10) were intraperitoneally injected with 1 × 106P. berghei ANKA-infected red blood cells, and the mice in the control group (n = 10) were intraperitoneally injected with a single dose of 0.85% normal saline. Kidney sections were collected and used for histopathological examination and the investigation of 4-hydroxynonenal (4-HNE) and heme oxygenase-1 (HO-1) expression through immunohistochemistry staining. The histopathology study revealed that the P. berghei-infected kidneys exhibited a greater area of tubular necrosis than those of the control group (p<0.05). The positive staining scores for 4-HNE and HO-1 expression in tubular epithelial cells of the P. berghei-infected group were significantly higher than those found for the control group (p<0.05). In addition, significant positive correlations were found between the tubular necrosis score and the positive staining scores for 4-HNE and HO-1 in the kidneys from the P. berghei-infected group. In conclusion, this finding demonstrates that increased expression of 4-HNE and HO-1 might be involved in the pathogenesis of acute tubular damage in the kidneys during malaria infection. Our results provide new insights into the pathogenesis of malaria-associated AKI and might provide guidelines for the future development of a therapeutic intervention for malaria