Nephroprotective effect of pioglitazone in a Wistar rat model of adenine­induced chronic kidney disease.

Chronic kidney disease (CKD) is a progressive disease with a high mortality rate and a worldwide prevalence of 13.4%, triggered by various diseases with high incidence. The aim of the present study was to investigate the anti-inflammatory and antifibrotic effect of pioglitazone on kidney in an adenine-induced Wistar rats and the mechanisms possibly involved. CKD was induced in 40 rats. Rats were divided into two groups, which were split into the following sub-groups: i) Therapeutic (pioglitazone administered after renal damage) divided into intact (healthy), adenine (CKD) and adenine/pioglitazone (treatment) and ii) prophylactic (adenine and pioglitazone administered at the same time) split into intact (healthy), adenine (CKD), endogenous reversion (recovery without treatment), adenine/pioglitazone (treatment) and pioglitazone sub-groups. Reverse transcription-quantitative PCR (collagen I, α-SMA and TGF-ß), and hematoxylin-eosin, Masson's trichrome and Sirius red staining were performed to measure histological markers of kidney damage, also the serum markers (urea, creatinine and uric acid) were performed, for analyze the effects of pioglitazone. In the adenine/pioglitazone rats of the therapeutic group, renal function parameters such as eGFR increased and serum creatinine decreased from those of untreated rats (CKD), however the renal index, serum urea, abnormalities in renal morphology, inflammatory cells and relative gene expression of collagen I, α-SMA and TGF-ß did not change relative to the CKD rats. In adenine/pioglitazone rats, extracellular matrix collagen accumulation was significantly lower than the CKD rats. On the other hand, in adenine/pioglitazone rats of the prophylactic group, the renal index, creatinine, urea, uric acid serum and relative gene expression of collagen I, α-SMA, and TGF-ß were significantly lower, as well as the presence of 2,8-dihydroxyadenine crystals, and extracellular matrix collagen compared with CKD rats. In addition, the eGFR in the treatment group was similar to healthy rats, renal morphology was restored, and inflammatory cells were significantly lower. In conclusion, pioglitazone has a nephroprotective effect when administered in the early stages of kidney damage, reducing inflammatory and fibrotic processes and improving glomerular filtration rate. Furthermore, in the late phase of treatment, a tendency to decrease creatinine and increase eGFR was observed.

Molecular and Antioxidant Characterization of Opuntia robusta Fruit Extract and Its Protective Effect against Diclofenac-Induced Acute Liver Injury in an In Vivo Rat Model.

A molecular characterization of the main phytochemicals and antioxidant activity of Opuntia robusta (OR) fruit extract was carried out, as well as an evaluation of its hepatoprotective effect against diclofenac (DF)-induced acute liver injury was evaluated. Phenols, flavonoids and betalains were quantified, and antioxidant characterization was performed by means of the ABTS•+, DPPH and FRAP assays. UPLC-QTOF-MS/MS was used to identify the main biocompounds present in OR fruit extract was carried out via. In the in vivo model, groups of rats were treated prophylactically with the OR fruit extract, betanin and N-acteylcysteine followed by a single dose of DF. Biochemical markers of oxidative stress (MDA and GSH) and relative gene expression of the inducible antioxidant response (Nrf2, Sod2, Hmox1, Nqo1 and Gclc), cell death (Casp3) and DNA repair (Gadd45a) were analyzed. Western blot analysis was performed to measure protein levels of Nrf2 and immunohistochemical analysis was used to assess caspase-3 activity in the experimental groups. In our study, the OR fruit extract showed strong antioxidant and cytoprotective capacity due to the presence of bioactive compounds, such as betalain and phenols. We conclude that OR fruit extract or selected components can be used clinically to support patients with acute liver injury.

Hepatoprotective Effect of Opuntia robusta Fruit Biocomponents in a Rat Model of Thioacetamide-Induced Liver Fibrosis.

Liver fibrosis is a chronic disease associated with oxidative stress that has a great impact on the population mortality. Due to their antioxidant capacity, we evaluated the protective effect of Opuntia robusta fruit (Or) on liver fibrosis. A nutraceutical characterization of Or was performed and a model of fibrosis was induced with thioacetamide (TAA) in Wistar rats. Aminotransferases, reduced glutathione (GSH) and histopathology were evaluated. Or contained 436.5 ± 57 mg of Betacyanins equivalents/L., 793 mg of catechin equivalents (CAE)/100 g for flavonoids, 1118 mg of gallic acid equivalents (GAE)/100 g for total phenols, 141.14 mg/100 g for vitamin C and 429.9 µg/100 g for vitamin E. The antioxidant capacity of Or was: 2.27 mmol of Trolox® equivalents (TE)/L (DPPH), 62.2 ± 5.0 µmol TE/g (ABTS•+), 80.2 ± 11.7 µmol TE/g (FRAP), 247.9 ± 15.6 µmol TE/g (AAPH) and 15.0% of H2O2 elimination. An increase (p < 0.05) of aminotransferases and a decrease (p < 0.05) of hepatic GSH was observed in the TAA group compared to the control and the concomitant groups. Histopathology showed changes in the normal architecture of the liver treated with TAA compared to the concomitant treatments. Or contains bioactive components with antioxidant capacity, which can reduce fibrotic liver damage.

Protective Effect of Curcumin against Doxazosin- and Carvedilol-Induced Oxidative Stress in HepG2 Cells.

Doxazosin and carvedilol have been evaluated as an alternative treatment against chronic liver lesions and for their possible role during the regeneration of damage caused by liver fibrosis in a hamster model. However, these drugs have been reported to induce morphological changes in hepatocytes, affecting the recovery of liver parenchyma. The effects of these α/𝛽 adrenoblockers on the viability of hepatocytes are unknown. Herein, we demonstrate the protective effect of curcumin against the possible side effects of doxazosin and carvedilol, drugs with proven antifibrotic activity. After pretreatment with 1 µM curcumin for 1 h, HepG2 cells were exposed to 0.1-25 µM doxazosin or carvedilol for 24, 48, and 72 h. Cell viability was assessed using the MTT assay and SYTOX green staining. Morphological changes were detected using the hematoxylin and eosin (H&E) staining and scanning electron microscopy (SEM). An expression of apoptotic and oxidative stress markers was analyzed using reverse transcription-quantitative PCR (RT-qPCR). The results indicate that doxazosin decreases cell viability in a time- and dose-dependent manner, whereas carvedilol increases cell proliferation; however, curcumin increases or maintains cell viability. SEM and H&E staining provided evidence that doxazosin and carvedilol induced morphological changes in HepG2 cells, and curcumin protected against these effects, maintaining the morphology in 90% of treated cells. Furthermore, curcumin positively regulated the expression of Nrf2, HO-1, and SOD1 mRNAs in cells treated with 0.1 and 0.5 µM doxazosin. Moreover, the Bcl-2/Bax ratio was higher in cells that were treated with curcumin before doxazosin or carvedilol. The present study demonstrates that curcumin controls doxazosin- and carvedilol-induced cytotoxicity and morphological changes in HepG2 cells possibly by overexpression of Nrf2.

Recovery from Liver Failure and Fibrosis in a Rat Portacaval Anastomosis Model after Neurointermediate Pituitary Lobectomy.

Liver diseases, including cirrhosis, viral hepatitis, and hepatocellular carcinoma, account for approximately two million annual deaths worldwide. They place a huge burden on the global healthcare systems, compelling researchers to find effective treatment for liver fibrosis-cirrhosis. Portacaval anastomosis (PCA) is a model of liver damage and fibrosis. Arginine vasopressin (AVP) has been implicated as a proinflammatory-profibrotic hormone. In rats, neurointermediate pituitary lobectomy (NIL) induces a permanent drop (80%) in AVP serum levels. We hypothesized that AVP deficiency (NIL-induced) may decrease liver damage and fibrosis in a rat PCA model. Male Wistar rats were divided into intact control (IC), NIL, PCA, and PCA+NIL groups. Liver function tests, liver gene relative expressions (IL-1, IL-10, TGF-ß, COLL-I, MMP-9, and MMP-13), and histopathological assessments were performed. In comparison with those in the IC and PCA groups, bilirubin, protein serum, and liver glycogen levels were restored in the PCA+NIL group. NIL in the PCA animals also decreased the gene expression levels of IL-1 and COLL-I, while increasing those of IL-10, TGF-ß, and MMP-13. Histopathology of this group also showed significantly decreased signs of liver damage with lower extent of collagen deposition and fibrosis. Low AVP serum levels were not enough to fully activate the AVP receptors resulting in the decreased activation of cell signaling pathways associated with proinflammatory-profibrotic responses, while activating cell molecular signaling pathways associated with an anti-inflammatory-fibrotic state. Thus, partial reversion of liver damage and fibrosis was observed. The study supports the crucial role of AVP in the inflammatory-fibrotic processes and maintenance of immune competence. The success of the AVP deficiency strategy suggests that blocking AVP receptors may be therapeutically useful to treat inflammatory-fibrotic liver diseases.

Glycomacropeptide Ameliorates Indomethacin-Induced Enteropathy in Rats by Modifying Intestinal Inflammation and Oxidative Stress.

Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy is considered a serious and increasing clinical problem without available treatment. Glycomacropeptide (GMP) is a 64-amino acid peptide derived from milk κ-casein with numerous biological activities. The aim of this study was to investigate the protective effect of GMP on NSAID enteropathy in rats. Enteropathy was induced by seven days oral indomethacin administration. Rats were orally GMP treated from seven days previous and during the establishment of the enteropathy model. Changes in metabolism, hematological and biochemical blood alterations, intestinal inflammation and oxidative damage were analyzed. Integrity barrier markers, macroscopic intestinal damage and survival rate were also evaluated. GMP treatment prevented anorexia and weight loss in animals. Furthermore, prophylaxis with GMP ameliorated the decline in hemoglobin, hematocrit, albumin and total protein levels. The treatment had no therapeutic efficacy on the decrease of occludin and mucin (MUC)-2 expression in intestinal tissue. However, GMP markedly decreased neutrophil infiltration, and CXCL1, interleukin-1ß and inducible nitric oxide synthase expression. Nitric oxide production and lipid hydroperoxide level in the small intestine were also diminished. These beneficial effects were mirrored by preventing ulcer development and increasing animal survival. These results suggest that GMP may protect against NSAID enteropathy through anti-inflammatory and antioxidant properties.

Curcumin and α/ß-Adrenergic Antagonists Cotreatment Reverse Liver Cirrhosis in Hamsters: Participation of Nrf-2 and NF-κB.

Liver cirrhosis is the result of an uncontrolled fibrogenetic process, due to the activation and subsequent differentiation into myofibroblasts of the hepatic stellate cells (HSC). It is known that HSC express adrenoreceptors (AR), and the use of AR antagonists protects experimental animals from cirrhosis. However, several studies suggest that the toxicity generated by metabolism of these antagonists would hinder its use in cirrhotic patients. In addition, liver fibrosis may be associated with a decrease of the antioxidant response of the nuclear factor erythroid 2-related factor 2 (Nrf-2) and the overregulation of the proinflammatory pathway of nuclear factor kappa B (NF-κB). Therefore, in the present work, the capacity of doxazosin (α1 antagonist), carvedilol (nonselective beta-adrenoceptor blocker with alpha 1-blocking properties), and curcumin (antioxidant and anti-inflammatory compound) to reverse liver cirrhosis and studying the possible modulation of Nrf-2 and NF-κB were evaluated. Hamsters received CCl4 for 20 weeks, and then treatments were immediately administered for 4 weeks more. The individual administration of doxazosin or carvedilol showed less ability to reverse cirrhosis in relation to concomitantly curcumin administration. However, the best effect was the combined effect of doxazosin, carvedilol, and curcumin, reversing liver fibrosis and decreasing the amount of collagen I (Sirius red stain) without affecting the morphology of hepatocytes (hematoxylin and eosin stain), showing normal hepatic function (glucose, albumin, AST, ALT, total bilirubin, and total proteins). In addition, carvedilol treatment and the combination of doxazosin with curcumin increased Nrf-2/NF-κB mRNA ratio and its protein expression in the inflammatory cells in the livers, possibly as another mechanism of hepatoprotection. Therefore, these results suggest for the first time that α/ß adrenergic blockers with curcumin completely reverse hepatic damage, possibly as a result of adrenergic antagonism on HSC and conceivably by the increase of Nrf-2/NF-κB mRNA ratio.

Extended low oxygen transmissibility contact lens use induces alterations in the concentration of proinflammatory cytokines, enzymes and electrolytes in tear fluid.

Prolonged and continuous use of contact lenses for as long as 3 or 4 weeks is common in Mexico due to the low socioeconomic status, poor patient education and self-neglect. Furthermore, wearing contact lenses with low oxygen permeability is common due to their low cost. Thus, patients seek ophthalmologic evaluation due to signs and symptoms of overuse such as red eye, discomfort and tearing. In the present study, the effect of wearing soft contact lenses with a low oxygen permeability on the tear fluid composition after 1 day, 1 week and 1 month without removing them was examined. In this prospective clinical trial, several tear fluid biomarkers were measured in 84 non-adapted contact lens wearers (NACLWs), including the pH, electrolytes, osmolarity, pro-inflammatory molecules [interleukin (IL)-8, IL-1ß and interferon (IFN)-γ], total protein (TP) levels and enzymes [aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and alkaline phosphatase (AP)]. The results indicated that the tear pH was significantly decreased after 1 day and 1 week; however, after 1 month of use, the tear pH level returned to the baseline. Tear electrolyte analysis demonstrated a significant decrease in Na+ at 1 day, 1 week and 1 month and Cl- levels at 1 week and 1 month, and a significant increase in Ca2+ at 1 week and 1 month, K+ at 1 day, 1 week and 1 month, IL-8 at 1 week and 1 month, IL-1ß only at 1 week and IFN-γ at 1 week and 1 month. Furthermore, the study observed an elevation of TP, AST, LDH and AP levels, however, there were no significant changes in ALT. In conclusion, the current study revealed that continuous wearing of soft contact lenses with low oxygen permeability increase tear fluid proinflammatory cytokine levels and enzymes reflecting tissue damage.

Doxazosin and Carvedilol Treatment Improves Hepatic Regeneration in a Hamster Model of Cirrhosis.

Regulation of the mechanisms of fibrosis is an important goal in the treatment of liver cirrhosis. One mechanism is the participation of hepatic stellate cells in fibrogenesis when activated by catecholamines. Consequently, α/ß adrenoblockers are proposed as an alternative treatment for chronic liver lesions such as fibrosis and/or cirrhosis and for possible liver regeneration. We herein analyzed the effect of doxazosin and carvedilol treatments during the regeneration of tissue in a hamster model of liver cirrhosis. Tissue samples were examined by H&E and PAS to evaluate tissue damage and with Sirius red to assess collagen fiber content. ALT, AST, albumin, and total proteins were examined by spectrophotometry. Determination of the levels of α-SMA and TGF-ß in hepatic tissue was examined by Western blot and of the expression of TIMP-2, MMP-13, α-FP, HGF, CK-7, and c-Myc was examined by qPCR. Treatment with doxazosin or carvedilol prompted histological recovery and reduced collagen fibers in the livers of cirrhotic hamsters. The expression of TIMP-2 decreased and that of MMP-13 increases in animals treated with adrenoblockers with respect to the group with cirrhosis. Additionally, the concentration of α-SMA and TGF-ß declined with both drugs with respect to placebo p<0.05. On the other hand, each drug treatment led to a distinct scenario for cell proliferation markers. Whereas doxazosin produced no irregularities in α-FP, Ki-67, and c-Myc expression, carvedilol induced an increment in the expression of these markers with respect to the intact. Hence, doxazosin and carvedilol are potential treatments for the regression of hepatic cirrhosis in hamsters in relation to the decrease of collagen in the hepatic parenchyma. However, at regeneration level we observed that doxazosin caused slight morphological changes in hepatocytes, such as its balonization without affecting the hepatic function, and on the other hand, carvedilol elicited a slight irregular expression of cell proliferation markers.

Vagotomy induces deregulation of the inflammatory response during the development of amoebic liver abscess in hamsters.

BACKGROUND: The parasympathetic nervous system modulates the immune response in the abdominal-pelvic gut through the vagus nerve, which releases acetylcholine. This endogenous ligand acts on α7 nicotinic receptors expressed on immune cells. OBJECTIVE: To study the mechanism of the production and regulation of cytokines in parasympathectomized and control hamsters during the development of amoebic liver abscesses (ALA) caused by Entamoeba histolytica. METHODOLOGY: Six- to 8-week-old male hamsters with and without vagotomy were used in a model of ALA. The animals were infected with trophozoites (350,000; HM1:IMSS strain) via the intrahepatic route and sacrificed at 6, 12, and 24 h and at 2, 4, and 7 days postinfection. Immune parameters were recorded at each time point using morphometric techniques including immunofluorescence and immunohistochemistry assays. These parameters included signal transducer and activator of transcription 3 (STAT3) levels, pro- and anti-inflammatory cytokine levels, and nuclear factor-κB (NFκB) activation in neutrophils and macrophages. RESULTS: Compared to the control groups, the vagotomized (VAG) hamsters showed a significant increase in NFκB activation in neutrophils and macrophages, and higher levels of interleukin (IL)-1ß, IL-6, interferon-γ, and tumor necrosis factor-α. VAG hamsters showed an increase in the expression of IL-8 and phosphorylated STAT3 during the first 24 h postinfection as well as slightly increased levels of transforming growth factor-ß on days 2-7 postinfection. No significant differences were demonstrated in the levels of IL-10. CONCLUSIONS: These results suggest that the vagus nerve plays an important role in the regulation of inflammation during ALA formation.