Cellular and molecular mechanisms of alpha lipoic acid’s protective effects against diclofenac-induced hepatorenal toxicity

Chemicals of an analytical grade were used in the current study. Diclofenac sodium, silymarin (SLY) and ALA (CAS No. 1077-28-7) were obtained from Novartis Pharma (Basel, Switzerland), SEDICO Pharmaceuticals (6th October City, Egypt) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Kits to measure the biochemical activity and oxidative stress parameters specific to aspartate aminotransferase (AST – cat. No. 260002), alkaline phosphatase (ALP – cat. No. AP 10 21), alanine aminotransferase (ALT – cat. No. 292002), total protein (cat. No. 310001), albumin (cat. No. 211001), uric acid (cat. No. UA 21 20), urea (cat. No. UR 21 10), total bilirubin (cat. No. BR 1110), creatinine (cat. No. CR 12 50), GSH (cat. No. GR 25 11), catalase enzyme (CAT – cat. No. CA 25 17) and malondialdehyde (MDA – cat. No. MD 25 29) were purchased from Bio Diagnostic (Dokki, Giza, Egypt). For anti-inflammatory activity assessment, Dulbecco’s modified Eagle’s medium (DMEM – Lonza, Basel, Switzerland), foetal bovine serum (FBS – Interpath, Somerton, VIC, Australia), penicillin and streptomycin (Sigma-Aldrich), glucose, L-glutamine and sodium pyruvate (Lonza), and murine RAW264.7 macrophage cells (American Type Culture Collection, Manassas, VA, USA) were obtained. For immunohistochemistry, rabbit polyclonal anti-caspase-3 (cat. No. A11319; ABclonal Biotech, Wuhan, China), horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit immunoglobulin G heavy and light chain (H&L) (cat. No. ab205718; Abcam, Cambridge, UK), streptavidin peroxidase (Thermo Scientific, Rockford, IL, USA) and 3,3'-diaminobenzidine tetra-hydrochloride (Sigma-Aldrich) were purchased.

Adult male Wistar rats aged 5 to 6 weeks and weighing between 130 and 160 g were sourced from the Animal House Colony at VacSera (Giza, Egypt). They were housed in the laboratory facilities at the Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Prior to the experiment, the rats were acclimatised for one week and were kept under standard laboratory conditions, including a controlled room temperature of 22 ± 1°C, relative humidity of 54–68% and a 12-h light/dark cycle. They had unrestricted access to a balanced diet consisting of 1% vitamin mixture, 4% mineral mixture, 10% corn oil, 20% sucrose, 0.2% cellulose, 10.5% casein and 54.3% starch, along with fresh water.

Thirty-five rats were randomly divided into 5 groups of seven rats each. Group I (negative control) rats received a daily oral dose of (1 mL) sterile distilled water for five days, followed by one intraperitoneal (I/P) sterile distilled water injection. Group II (DIC, positive control) rats received a daily oral dose of 1 mL distilled water for five days and were then subjected to a hepatorenal toxic insult through I/P injection of DIC in sterile distilled water at a dose of 50 (mg/kg body weight (b.w.)) 1 h after the last oral dose (31). Group III (SLY) rats were pretreated orally with SLY at a dose of 100 mg/kg b.w. daily as a standard hepatoprotective reference drug for five days and were then subjected to the same insult as previously described, also 1 h after their SLY dose (21). Group IV (ALA 50) rats were pretreated orally with ALA at a dose of 50 mg/kg b.w. daily for five days before hepatotoxicity induction as in the other groups (25). Group V (ALA 100) differed only from the ALA 50 group in the dose which they received, this being 100 mg/kg b.w. (12).

At the end of the experiment, the animals were anaesthetised with I/P ketamine at 91 mg/kg b.w. 24 h after the last treatment. Collection of blood samples from the retro-orbital sinuses and their transfer to gel separator tubes to collect serum samples followed. The serum was collected by centrifugation of the blood at 3,000 × g at 4°C for 10 min and stored at –80°C to further determine biochemical and antioxidant biomarkers. The animals were weighed and euthanised by cervical dislocation. The liver and kidney tissues were removed, excess fat was trimmed, and then they were weighed. The tissues were divided into three portions, with two sets being stored at –80°C for quantitative reverse-transcription PCR (qRT-PCR) and antioxidant analysis. The final set of hepatorenal tissues was preserved in 10% neutral-buffered formalin for histological and immunohistochemical examination.

Murine RAW264.7 macrophage cells were cultured in DMEM, which included 4.5 g/L of glucose, L-glutamine and sodium pyruvate, and had 5% FBS and 100 U/mL of penicillin and streptomycin added to it. The cells were maintained at 37°C in a 5% CO₂ atmosphere until they achieved 90% confluency. At a seeding density of 0.85 × 106 mL⁻¹, the cells were divided and plated into 96-well cell culture plates. They were then incubated for 24 h, rinsed with serum-free media and exposed to several treatments that had been researched, guided by prior research that demonstrated broad-spectrum anti-inflammatory and antioxidant effects of natural products, supporting the rationale for investigating similar activities of alpha-lipoic acid and diclofenac in this study. (9).

Lipopolysaccharides (LPS) at a concentration of 50 ng/mL stimulated RAW264.7 macrophage cells to generate nitric oxide. The NO was quantified as total nitrite content using freshly made Griess reagent as described previously (9, 22). In summary, after administering ALA at concentrations of 50, 25, 12.5, 6.25, 3.125, and 1.5625 μg/mL for 2 h, an inflammatory response was induced by the addition of lipopolysaccharide (LPS) at 50 ng/mL. The cells were then incubated for a further 24 h to evaluate the anti-inflammatory effects of ALA pre-treatment. A microplate spectrophotometer (CLARIOstar; BMG Labtech, Ortenberg, Germany) was employed to measure absorbance at 540 nm after 100 μL of the supernatant was collected and mixed with Griess reagents. The viability of the treated RAW264.7 macrophage cells was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment and stimulation, the cells were co-incubated with MTT (0.12 mg/mL) for 4 h. Following the removal of the supernatant, the optical density was measured at 510 nm using the same microplate spectrophotometer. Cells which had not been stimulated by LPS (positive control cells) were used as a baseline for calculating the percentage of viable cells and nitric oxide (NO) compared with the negative control (untreated and unstimulated cells).

The ROS assay was carried out on the same murine RAW264.7 macrophage cells used in the previous stages. It utilised a 2′,7′-dichlorofluorescein diacetate cellular ROS detection assay kit (cat No. ab113851; Abcam, Cambridge, UK), in accordance with the manufacturer’s guidelines and as described in our earlier studies. A positive control of 250 μM tert-butyl hydroperoxide was used. The fold increase in ROS generation was determined relative to the untreated control (cells treated with the buffer as specified in the procedure) (7).

Liver function was assessed via measurement of ALT, AST, ALP, total proteins, albumin and total bilirubin. Kidney function was also evaluated through the measurement of urea, creatinine and uric acid. All measurements were made with standardised kits purchased from Bio Diagnostic and used as directed in the manufacturer’s protocol.

Hepatic and renal tissues were mixed uniformly in cold buffered saline (10 mL/g tissue) with a glass homogeniser before being centrifuged at 4,000 rpm for 15 min at 4°C. The buffer for measuring MDA contained 50 mM potassium phosphate at pH 7.5, as did the buffer for GSH along with 1 mM ethylenediaminetetraacetic acid (EDTA). For CAT analysis, the buffer was composed of 50 mM potassium phosphate at pH 7.4, 1 mM EDTA and 1 mL/L Triton X-100 per gram of tissue. The concentrations of MDA, GSH and CAT were then assessed in the supernatants according to standard methods (3, 14).

Livers and kidneys from different experimental groups were collected, preserved in 10% neutral-buffered formalin for 48 h, embedded in paraffin blocks, sliced into sections and stained with haematoxylin and eosin. Two veterinary pathologists rated sections blindly semi-quantitatively for hepatic (neuroinflammatory) and renal (glomerular, interstitial and vascular) abnormalities. The grading of the hepatic and renal lesions was performed applying previously described techniques (22).

The tissue sections were dewaxed and rehydrated before their treatment with 10 mM citrate buffer for antigen retrieval. In a humidified chamber, the primary antibody, rabbit polyclonal antibody against caspase-3, was incubated in sections overnight at 4°C. The tissue sections were then treated for 10 min with goat anti-rabbit IgG H&L (HRP). Slides were treated with streptavidin peroxidase and 3,3'-diaminobenzidine tetra-hydrochloride and were then incubated for 10 min. After counterstaining with Mayer’s haematoxylin, the slides were dehydrated and mounted.

The primary antibodies were omitted in negative controls (27). A Qwin 500 Image Analyzer (Leica Microsystems, Wetzlar, Germany) was used to examine tissue sections.

The immunolabelled regions were determined in each field of stained sections. The positively stained area percentage (%) was measured by counting positive cells and the total number of cells was counted under a high-power field (×400) microscope in 10 microscopic fields/slides that were randomly selected.

Messenger RNA (mRNA) expression levels of Kelch-like erythroid cell-derived protein with cap 'n' collar homology– associated protein 1 (Keap1)/nuclear erythroid 2-related factor 2 (Nrf2) signalling pathway–related genes were determined in the hepatic and renal tissues using a qRT-PCR. Total cellular RNA from fresh liver and kidney tissue samples (approximately 100 mg) was isolated in ice-cold TRIZOL reagent according to the manufacturer’s technique (Invitrogen, San Diego, CA, USA). The yield and purity of the RNA were determined using a NanoDrop System (Thermo Fisher Scientific, Wilmington, DE, USA) at 260/280 nm wavelengths. A RevertAid cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania) was used with 1 μg RNA samples to reverse-transcribe them into cDNA. The relative mRNA expression levels of Nrf2, haem oxygenase 1 (HO-1) and nicotinamide adenine (phosphate) reduced form : quinone oxidoreductase 1 (NQO1) were determined using an ABI 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The β-actin gene was the housekeeping gene. The oligonucleotide primers employed for qRT-PCR analyses are recorded in Table 1. Relative gene expression was calculated as fold-change difference after normalisation against the mRNA level of β-actin using the threshold-cycle formula (2^–△△CT).

The various analytical measurements were performed in triplicate, and the results were reported as the mean ± standard deviation (SD), where n = 7. GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA, USA) was used to determine the half maximal inhibitory concentration (IC₅₀) and half maximal effective concentration of the tested drugs, which involved performing a nonlinear regression of the log-transformed concentration’s variable slope (four parameters) against the normalised response. Mean difference values in the experiments at P-value < 0.05 were considered statistically significant. One-way or two-way analysis of variance was performed to compare the means using post hoc Tukey’s corrections for multiple comparisons or a Kruskal–Wallis test followed by a Dunn’s test for the histopathological scoring, or a Tukey multiple-comparisons test for all other experiments at 95% confidence.

The anti-inflammatory activity of ALA at 50ug/mL and lower concentrations in six different 1 : 2 serially dilutions against the effect of DIC relative to the control was assessed using a nitric oxide assay (Fig. 1A). Inhibition of NO production by 50% was observed at 7.8 ± 1.01 and 22.64 ± 1.4 μg/mL (mean ± SD) for ALA and DIC, respectively. A dose-dependent decline in NO production was exhibited for ALA and DIC with a significant difference (P-value < 0.05) relative to the control down to 6.25 and 25 μg/mL, respectively (Fig. 1A). The different concentrations showed cell viability above 90% relative to the negative control as measured by the MTT assay, indicating that the NO-depletive activity is related to the drug treatment rather than the cytotoxicity (Fig. 1B).

Fig. 1.

The inhibitory effect on murine RAW264.7 macrophage cells of different α-lipoic acid and diclofenac sodium concentrations on A) nitric oxide (NO) production and B) cell viability assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (n = 3). Data was expressed as mean ± standard deviation. IC₅₀ – half maximal inhibitory concentration; ** and **** – significant difference relative to the negative control at P-value < 0.01 and P-value < 0.0001, respectively

Tert-butyl hydroperoxide at 50 μM increased the ROS production to 175.25 ± 8.28% (Fig. 2). Alpha lipoic acid produced a significant dosedependent inhibition of ROS production at 50, 25, 12.5 and 6.25 μg/mL concentrations relative to the control, with IC₅₀ of 6.279 ± 1.58 μg/mL (Fig. 2).

Fig. 2.

The inhibitory effect in murine RAW264.7 macrophage cells of different alpha lipoic acid (ALA) and diclofenac sodium concentrations on reactive oxygen species (ROS) production. Data are expressed as mean ± standard deviation, n = 3. EC₅₀ – half maximal effective concentration; TBHP – tert-butyl hydroperoxide; ** and *** – significant differences at P-value ≤ 0.005 and 0.0005, respectively

Liver function markers (ALT, AST, ALP and total bilirubin) and kidney function markers (urea, creatinine and uric acid) were significantly elevated in the DIC-challenged and hepatorenally unprotected group relative to the negative control group. Serum total proteins, albumin and globulin concentrations showed a significant reduction in the DIC-challenged and hepatorenally unprotected group compared with the negative control group. The pre-administration of SLY, ALA at 50 mg/kg b.w. and ALA at 100 mg/kg b.w. revealed a substantial protective effect, and less damage from the insult than in the unprotected DIC group. This protection was revealed through a substantial inhibition of liver enzymes (AST, ALT and ALP), total bilirubin and kidney function markers (urea, creatinine, and uric acid), and elevation of total protein, albumin and globulin concentrations in comparison with the DIC-challenged group. The group receiving the higher dose, ALA 100, showed the best improvement when compared with the other pretreated groups (Table 2).

Fig. 3.

The effect of hepatoprotective compounds on hepatic and renal oxidative biomarkers in Wistar rats subjected to hepatorenal toxic insult by diclofenac sodium (DIC) and contrast in rats denied the compounds. A and D – effect on malondialdehyde (MDA); B and E – effect on catalase (CAT); C and F – effect on reduced glutathione (GSH). Data are expressed as mean ± standard deviation. n = 7 rats/group. * and ** – significant difference vs negative control group at P-value ≤ 0.05 and P-value ≤ 0.005, respectively; ^# and ^## – significant difference vs DIC group at P-value ≤ 0.05 and P-value ≤ 0.005, respectively; SLY – silymarin at 100 mg/kg body weight (b.w.); ALA 50 – α-lipoic acid at 50 mg/kg b.w.; ALA 100 – α-lipoic acid at 100 mg/kg b.w.

The activity of MDA and CAT was significantly elevated, but GSH was depleted in the DIC group’s hepatic and renal tissues, in contrast to the negative control group. Interestingly, the pretreatment with SLY at 100 mg/kg b.w., ALA at 50 mg/kg b.w. and ALA at 100 mg/kg b.w. revealed a significant inhibition of MDA and CAT activity in addition to GSH elevation, which contrasted with the DIC-challenged and hepatorenally unprotected group. Furthermore, the group that received the higher dose of ALA showed the best improvement when compared with the other pretreated groups.

Haematoxylin and eosin staining and light microscopy were utilised to demonstrate structural histopathological changes in the liver and kidney tissues in various groups. The evaluation of liver tissues from the negative control group showed normal histological architecture (Fig. 4A). The DIC-challenged and hepatorenally unprotected rats exhibited significant histopathological changes, including areas of hydropic degeneration (ballooning) and fatty change (steatosis), along with hepatocellular cytoplasmic microvacuolation, apoptosis, hepatocellular necrosis and mononuclear cell infiltration in central areas (Fig. 4B). Silymarin-treated rats showed signs of hepatocellular regeneration, mild vacuolar degeneration and sporadic hepatocellular necrosis (Fig. 4C). Histopathological investigation of the hepatic tissues from the ALA 50 and ALA 100 groups showed fewer histopathological hepatic alterations (Fig. 4D and E). In the ALA 50 rats, the hepatocytes exhibited moderate vacuolar degeneration, distension from fat vacuoles and congestion of the central vein. The liver samples of rats pretreated with ALA at 100 mg/kg b.w. were free of the previously described hepatic lesions found in DIC-treated and unprotected rats, and cellular regeneration was seen along with a milder degree of cellular necrosis and fatty changes. The scores for histopathological hepatic lesions, including necro-inflammatory changes, are summarised in Fig. 6A, with all treated groups compared with the negative control group. Additionally, the SLY, ALA 50 and ALA 100 groups were compared with the DIC-challenged and hepatorenally unprotected group.

Fig. 4.

Photomicrographs of haematoxylin and eosin–stained sections of the liver in different experimental groups of Wistar rats subjected to hepatorenal toxic insult by diclofenac sodium (DIC) after being administered or denied protective compounds. A – the liver of a negative control group rat showing normal histological architecture of liver tissue. B – the liver of a DIC-treated and hepatorenally unprotected group rat showing diffused hydropic degeneration of hepatocytes, apoptosis, hepatocellular necrosis and mononuclear infiltration. C – the liver of a silymarin-treated group rat dosed at 100 mg/kg body weight (b.w.) showing hepatocellular regeneration and mild vacuolar degeneration. D – the liver of an α-lipoic acid (ALA)-treated group rat dosed at 50 mg/kg b.w. showing moderate vacuolar degeneration. E – the liver of an ALA-treated group rat dosed at 100 mg/kg b.w. showing marked hepatocellular regeneration, mild cellular necrosis and fatty changes

The kidney histopathological investigations of the negative control group revealed normal architecture with normal glomerulus and tubular arrangement of the renal tissue (Fig. 5A). The DIC-challenged and hepatorenally unprotected rats revealed dilatation and renal vessels congestion, renal glomerular distortion with vacuolation in the endothelial lining of the glomerular tuft, increased mononuclear cell infiltration of the glomerulus and severe and extensive renal tubular necrosis, with subsequent loss of cellular constituents and desquamation of tubular cells as well as formation of intraluminal eosinophilic cast and severe fatty change in the epithelial lining of renal tubules (Fig. 5B). Silymarin-treated rats showed renal vascular dilation and congestion as well as cellular desquamation and intraluminal cast formation in the tubular cells (Fig. 5C). Histological examinations of the kidneys from ALA 50 and ALA 100 pretreated rats showed none of the histopathological renal alteration; the histological structure of the endothelial cell lining of the glomerular tuft was normal and renal necrosis was less (Fig. 5D and E). The histopathological renal lesion scores, including for glomerular, interstitial and vascular changes, are summarised in Fig. 6B. All the pretreated groups were compared with the negative control group. Additionally, the SLY, ALA 50 and ALA 100 groups were compared with the DIC-challenged and hepatorenally unprotected group.

Fig. 5.

Photomicrographs of haematoxylin and eosin–stained sections of the kidney in different experimental groups of Wistar rats subjected to hepatorenal toxic insult by diclofenac sodium (DIC) after being administered or denied protective compounds. A – the kidney of a negative control group rat showing normal renal architecture with a normal glomerulus and renal tubule arrangement. B – the kidney of a DIC-treated and hepatorenally unprotected group rat showing vacuolisation in the endothelial lining of the glomerular tuft, congestion of renal vessels, desquamation of tubular cells with intraluminal eosinophilic cast formation, severe fatty change in the epithelial lining of renal tubules and necrosis of renal tubules. C – the kidney of a silymarin-treated group rat dosed at 100 mg/kg body weight (b.w.) showing dilation and congestion of renal vessels, tubular cell desquamation and intraluminal cast formation. D and E – the kidneys of α-lipoic acid–treated group rats dosed at 50 mg/kg b.w. or 100 mg/kg b.w. showing marked improvement in renal alterations, restored normal histological structure of the endothelial cell lining of the glomerular tuft and reduced renal necrosis

Fig. 6.

Histopathological evaluation of hepatic and renal lesion scoring for Wistar rat organs after hepatorenal toxic insult with diclofenac sodium (DIC) preceded by administration of protective compounds and contrast in the organs of rats denied the compounds. A – necro-inflammatory score in liver tissue. B – glomerular, interstitial and vascular score in kidney tissue. Data are expressed as median and interquartile range. Adjusted P values were considered significant at P-value ≤ 0.05. DIC – scores for rats administered DIC and not protected hepatorenally; SLY – scores for rats administered silymarin at 100 mg/kg body weight (b.w.) as hepatorenal protection, ALA 50 – scores for rats administered α-lipoic acid at 50 mg/kg b.w. as hepatorenal protection; ALA 100 – scores for rats administered α-lipoic acid at 100 mg/kg b.w. as hepatorenal protection; * and ** – significant difference vs normal control group at P-value ≤ 0.05 and P-value ≤ 0.005, respectively; # and ## – significance difference vs DIC group at P-value ≤ 0.05 and P-value ≤ 0.005, respectively

Immunohistochemical staining was employed to evaluate the expression of liver caspase-3 protein in all the experimental groups. Caspase-3 immunostaining was not detected in the liver tissue in the negative control group (Fig. 7A). The slides of the DIC-challenged and hepatorenally unprotected group revealed strong positive immune expression of caspase-3 protein in the hepatocytes undergoing apoptosis, in contrast to the negative control group (Fig. 7B). The SLY group revealed moderately caspase-3-stained hepatocytes (Fig. 7C). The ALA 50 group showed moderate caspase-3 immunostaining of hepatocytes, and the ALA 100 group showed a reduction of immunopositive cells (Fig. 7D and E). Image analysis of the DIC-treated and unprotected group showed a significant upregulation in caspase-3 immune expression compared with the negative control group (Fig. 9A), which was significantly reversed in the groups pre-treated with either dose of ALA or with SLY. A significant dose-dependent decline in the caspase-3 staining percentage was indicated, comparing ALA 50 with ALA 100 (P-value = 0.0037). Notably, the ALA at dose of 100 mg/kg dose was not statistically different compared with the control, unlike the lower 50 mg/kg dose (Fig. 9A).

Fig. 7.

Photomicrographs of caspase-3 activity in livers in different experimental groups of Wistar rats subjected to hepatorenal toxic insult by diclofenac sodium (DIC) after being administered or denied protective compounds. A – the liver of a normal control group rat showing no detectable caspase-3 immunostaining. B – the liver of a DIC-treated and hepatorenally unprotected group rat showing strong caspase-3 immunostaining of hepatocytes undergoing apoptosis. C – the liver of a silymarin-treated group rat dosed at 100 mg/kg body weight (b.w.) showing moderately stained hepatocytes. D – the liver of an α-lipoic acid–treated group rat dosed at 50 mg/kg b.w. showing moderate caspase-3 immunostaining of hepatocytes. E – the liver of an α-lipoic acid–treated group rat dosed at 100 mg/kg b.w. showing a reduction of the immunopositive cells

The immunohistochemical staining revealed that no detectable expression of caspase-3 protein was recorded in the renal corpuscles or tubules of the negative control group (Fig. 8A). Immunohistochemical staining of the DIC-treated and hepatorenally unprotected group kidneys’ slides showed intense caspase-3 staining in the glomeruli, proximal and distal renal tubules, which contrasted with the absence of this in the negative control group (Fig. 8B). Pretreatment with SLY showed that caspase-3 staining of glomeruli and tubules (Fig. 8C) was not statistically significantly more intense than that in the DIC-treated group (Fig. 9B). The results for the ALA 50 and ALA 100 pretreated groups indicated significantly reduced caspase-3 activity, and low caspase-3 expression was recorded in the glomeruli and tubules of both groups (Figs 8D and E, and 9B). Image analysis to evaluate the area corresponding to caspase-3 expression in the DIC-challenged and hepatorenally unprotected group found this expression to be significantly higher in comparison with the negative control group (Fig. 9B). Caspase-3 expression was significantly less in the groups given ALA pretreatment, with no significant difference between the 50 and 100 mg/kg b.w. doses of ALA.

Fig. 8.

Photomicrographs of caspase-3 activity in kidney in the different experimental groups of Wistar rats subjected to hepatorenal toxic insult by diclofenac sodium (DIC) after being administered or denied protective compounds. A – the kidney of a normal control group rat showing no expression of caspase-3 in the renal corpuscles and tubules. B – the kidney of a DIC-treated and hepatorenally unprotected group rat showing strong positive caspase-3 expression in the glomeruli and tubules. C – the kidney of a silymarin-treated group rat dosed at 100 mg/kg body weight (b.w.) showing moderately stained glomeruli and tubules. D and E – the kidneys of α-lipoic acid–treated group rats dosed at 50 mg/kg b.w. or 100 mg/kg b.w. showing a reduction of the immunopositive cells

Fig. 9.

Image analysis data to evaluate the area corresponding to caspase-3 protein expression in the organs of different experimental groups of Wistar rats subjected to hepatorenal toxic insult by diclofenac sodium (DIC) after being administered or denied protective compounds. A – caspase-3 immunostaining expressed as area % in liver tissues. B – caspase-3 immunostaining expressed as area % in kidney tissues. Data are expressed as mean +/− standard deviation. DIC – diclofenac; SLY – silymarin at 100 mg/kg body weight (b.w.); ALA 50 – α-lipoic acid at 50 mg/kg b.w.; ALA-100 – α-lipoic acid at 100 mg/kg b.w.; * and ** – significant difference vs normal control group at P-value ≤ 0.05 and P-value ≤ 0.005, respectively; ## – significant difference vs DIC group at P-value ≤ 0.005

To further evaluate the mechanism of the protective effects of ALA against DIC-induced hepatorenal toxicity, the primary mRNA species involved in the Nrf2 signalling pathway were assessed by qRT-PCR. At transcriptional levels, DIC-intoxicated and unprotected rats showed a significant downregulation in Nrf2, NQO-1 and HO-1 compared with the normal control group. By contrast, supplementation with ALA 50 and ALA 100 upregulated the transcriptional levels of Nrf2, NQO-1 and HO-1 in both liver (Fig. 10A–C) and kidney (Fig. 10D–F), doing so statistically significantly more intensely in most cases compared with these genes’ transcriptional levels in the DIC group.

Fig. 10.

Alpha-lipoic acid (ALA) effects on the messenger RNA expression levels of genes in the liver and kidney tissues of Wistar rats subjected to hepatorenal toxic insult by diclofenac sodium (DIC) after being administered or denied protective compounds. A and D – effect on nuclear factor erythroid 2-related factor 2 (Nrf2). B and E – effect on nicotinamide adenine (phosphate) reduced form : quinone oxidoreductase 1 (NQO1). C and F – effect on haem oxygenase 1 (HO-1). Data are expressed as mean ± standard deviation. n = 7 rats/group; SLY – silymarin at 100 mg/kg body weight (b.w.); ALA 50 – α-lipoic acid at 50 mg/kg b.w.; ALA 100 – α-lipoic acid at 100 mg/kg b.w.; * and ** significant difference vs normal control group at P-value ≤ 0.05 and P-value ≤ 0.005, respectively; # – significant difference vs DIC group at P-value ≤ 0.05