Hepatitis results in high morbidity and mortality rates in horses,1,2 and investigation of new therapeutic agents is needed. Oxidative injury is a prominent mechanism of hepatic injury, and the positive effects of the milk thistle derivative silibinin on antioxidant capacity, as well as its direct oxidant-scavenging ability, are important features in its hepatoprotective actions.3 The antioxidant properties of silibinin are dose dependent in mice.4 However, the effects of silibinin administration on antioxidant capacity in horses are unknown. Multiple assays for enzymes and substrates can be performed to evaluate antioxidant capacity in vivo. Estimation of the plasma ORAC provides information on resistance to peroxyl radical injury5 and has been used to estimate antioxidant capacity in horses.6 The GPOX enzyme is a potent detoxifier of superoxide anions and hydrogen peroxide and in this manner limits oxidative injury.7 Another critical enzyme in oxidant protection, NQO1, functions to catalyze the 2-electron reduction of quinones to hydroquinones in cells, limiting 1-electron reduction, which results in formation of reactive oxygen species.8 Glutathione is the major intracellular reducing agent, functioning to protect against oxidative stress.9 Shifting of GSH to GSSG indicates a shift of redox status toward oxidative stress.10 Maximizing antioxidant capacity is protective against oxidative stress and injury from oxidative processes.
Amplification of inflammation caused by potent cytokines released by myeloid cells, such as TNFα and IL-1, promotes a cascade of events that results in sequestration of polymorphonuclear leukocytes in tissues and parenchymal dysfunction.11 Inflammatory responses are beneficial in recognition of foreign antigens and clearing infection. However, prolonged or overexuberant expression of inflammatory cytokines can be detrimental and has been linked to pathological changes associated with disease.12 Hepatocellular injury may be caused by a combination of the primary oxidative effect and the secondary inflammatory response induced by damaged hepatocytes.13 Tumor necrosis factor α is directly toxic to hepatocytes and induces apoptosis.14 Interleukin-1β and IL-6 reduce hepatocyte protein synthesis, carbohydrate metabolism, and cytochrome P450-dependent detoxification.15 Blood chemokine concentration has been associated with severity of hepatic disease in humans.16 Derangement in the balance of pro- and anti-inflammatory serum cytokines is characteristic of alcoholic cirrhosis and is predictive of prognosis and mortality in humans.17 Inhibiting cytokine release and subsequent inflammatory cell recruitment may limit organ damage. Silibinin protects against inflammation by limiting oxidative injury, inhibiting neutrophil migration, and regulating inflammatory mediators in rats.11 Silibinin inhibits expression and synthesis of inflammatory cytokines TNFα, IL-1, and IL-2 in the presence of diseases and inflammatory stimuli in mice.18,19
The purpose of the study reported here was to evaluate antioxidant capacity and inflammatory cytokine gene expression in horses fed silibinin complexed with phospholipid. It was hypothesized that oral silibinin administration would increase antioxidant capacity in the blood of healthy horses, that these effects would be dose dependent, and that oral silibinin phospholipid administration would not alter gene expression of inflammatory cytokines in the blood of healthy horses because of the absence of preexisting disease or inflammation.
Materials and Methods
This study was performed in conjunction with an institutional animal care and use committee-approved phase II pharmacokinetic study.20 Five horses owned by the Colorado State University Veterinary Teaching Hospital and acclimatized to their housing were used with permission, and environmental conditions were not changed. Horses were group housed in a paddock without access to grass. Horses received water ad libitum and Timothy grass hay once daily, providing for consumption of approximately 12 kg of hay/horse per day. All horses were geldings with a mean ± SD age of 13 ± 5 years (median, 14 years [range, 5 to 17 years]) and mean weight of 582 ± SD 65 kg (median, 615 kg [range, 472 to 625 kg]). There were 3 Quarter Horses, 1 Arabian, and 1 Andalusian. Horses were screened prior to inclusion in the study for evidence of gastrointestinal tract or liver dysfunction by use of physical examination and serum biochemical analyses and were selected only if they readily consumed the carrier diet (400 g of pelleted feed,a 50 g of wheat bran, and 150 mL of water [per meal]). Body weight was measured with a commercial scale, and signalment was recorded. Horses consumed each dose twice daily each day for 7 days during 4 administration periods, with progressively higher doses of silibinin phospholipidb administered during each period and with each period separated by a washout period (minimum of 2 weeks). During week 1, twice daily, horses consumed the carrier diet without silibinin phospholipid. During week 2, horses were fed the diet plus 20 mg of silibinin phospholipid/kg of carrier diet, resulting in a 6.5 mg/kg of body weight dose of silibinin. During week 3, horses were fed the diet plus 40 mg of silibinin phospholipid/kg of diet, resulting in a 13 mg/kg dose of silibinin. During week 4, horses were fed the diet plus 80 mg of silibinin phospholipid/kg of diet, resulting in a 26 mg/kg dose of silibinin. Mean ± SD total dose of silibinin phospholipid administered in feed in week 2 was 11.6 ± 1.3 g (median, 12.3 [range, 9.4 to 12.5 g]), in week 3 was 23.3 ± 2.6 g (median, 24.6 g [range, 18.9 to 25.0 g], and week 4 was 46.5 ± 5.2 g (median, 49.2 g [range, 37.8 to 50.0 g]). All 5 horses received identical treatments, except on day 7 of week 4 (highest-dose week), when the final meal supplemented with silibinin phospholipid was incompletely consumed (1 horse) or consumed slowly (1 horse).
Sample collection—Blood samples (total volume, 40 mL) were obtained from horses on day 1, prior to administration of unsupplemented diet or carrier diet mixed with silibinin phospholipid, and on day 7, 1 hour following the final meal of the study diet, each week. Blood for antioxidant analysis was collected directly into EDTA tubes. Plasma was immediately separated via centrifugation at 2,500 × g for 10 minutes, transferred to cryovials, submerged in liquid nitrogen until frozen, and stored at −80°C until analysis. Following plasma removal, the buffy coat layer was transferred to a 15-mL tube, and the pelleted RBCs were transferred to a cryovial, submerged in liquid nitrogen until frozen, and stored at −80°C until analysis. The buffy coat sample was brought to a final 6-mL sample volume with PBS solution, layered onto 4 mL of a mixture of nonionic, synthetic polymer of sucrosec and sodium diatrizoate,d and centrifuged at 800 × g for 30 minutes at 20°C. Following centrifugation, the fraction containing PBMCs was collected and washed twice with PBS solution. The PBMCs were counted with a hemacytometer and resuspended in PBS solution prior to freezing at −80°C until analysis. Blood for cytokine gene expression analysis was collected directly into commercially available evacuated tubes containing a proprietary additive to stabilize the in vivo gene transcription profile by reduction of in vitro RNA degradation,e maintained at 20°C for 60 minutes, and then frozen at −20°C until analysis as per the manufacturer's instructions.
Protein assay—Protein measurement in plasma, RBC, and PBMC samples was necessary to report protein-corrected values of glutathione and antioxidant enzymes. The RBC and PBMC samples required additional processing prior to analysis and were thawed on ice and diluted in 25mM Tris (pH, 7.4). Samples were then disrupted by sonicationf in three 2-second bursts at 30% power on ice and centrifuged at 15,000 × g; the supernatant was collected for further analysis. Colorimetric measurement of protein was performed by use of bicinchoninic acid and standard curves of bovine serum albumin.g Samples were added to a 96-well microplate with 200 μL of working reagent containing bicinchoninic acid, and incubated for 30 minutes at 37°C. Following incubation, absorbance was measured at 562 nm with a microplate reader.h Net absorbance was calculated by subtracting values of blank samples from values of bovine albumin standards and test sample replicates. Standard curves were graphed by plotting mean blank-corrected albumin standard values versus concentration and test samples estimated via linear regression. Protein concentration was reported in milligrams per milliliter.
ORAC—The ORAC of plasma was measured with a commercially available assayi as described by Ungvari et al.21 The assay evaluated the ability of plasma samples to delay oxidation of a fluorescent probe by peroxyl radicals relative to known concentrations of a water-soluble vitamin E analog22 and has been validated in horses and other species.23 Plasma samples were thawed on ice, vortexed, and diluted 1:100. Fluorescein solutionj and either plasma or standard curve samples were added to a 96-well microtiter plate and incubated for 30 minutes at 37°C. The plate was read immediately following addition of 2,2′-azobis(2-methylpropionamidine) hydrochloride.k Fluorescence was recorded every 84 seconds for 1 hour with a fluorescent microplate readerh with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Net AUC for plasma samples was calculated by subtracting blank sample AUC from test sample AUC and then compared with an antioxidant standard curve of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acidl constructed by graphing the net AUC of vitamin E analog samples of known concentrations. Lower limit of quantitation was 2.5μM vitamin E analog. Linear regression was used to estimate the vitamin E analog equivalents of plasma samples on the basis of the vitamin E analog standard curve. Plasma ORAC was compared among horses that were administered different doses of silibinin phospholipid.
NQO1 activity—The NQO1 of PBMC and RBC samples was measured by use of the method described by Gustafson et al.24 The PBMC and RBC lysates were thawed on ice. A 25mM Tris plus 0.7% bovine serum albumin solution was added to a methacrylate cuvette, followed by DCPIPm (40μM final solution) and nicotinamide adenine dinucleotiden (200μM final solution). Samples were evaluated in duplicate and mixed immediately prior to measurement of absorbance at 600 nm for 120 seconds via spectrophotometer,o with and without the addition of dicumarol (20μM final solution). The NQO1 activity was defined as the dicumarol-inhibited decrease in absorbance at 600 nm, or the difference in the change in optical density per minute between dicumarol-negative and dicumarol-positive samples. The NQO1 activity was converted to nmol of DCPIP reduced/min through calculations that used the extinction coefficient of DCPIP (21mM−1cm−1). The NQO1 was normalized for protein content and expressed as nmol of DCPIP/min/mg.
GPOX activity—The GPOX activity of PBMC and RBC samples was measured by use of the method described by Gustafson et al.25 A GPOX assay has been validated in horses.6 The reaction mixture consisted of 2.59 mL of 50mM potassium phosphate buffer (pH, 7.0) with 1mM EDTA, 10 μL of 5mM sodium azide, 100 μL of 150mM GSH, 100 μL of 2.2mM hydrogen peroxide, 5 μL of glutathione reductase, and 100 μL of 8.4mM NADPH in a methacrylate cuvette. Test samples were added after a linear rate was established at 340 nm absorbance for 240 seconds in a spectrophotometer.o The GPOX activity was defined as the rate of NADPH oxidation in the presence of glutathione and glutathione reductase, or the difference in linear rates following sample addition. The GPOX activity was converted to pmol of NADPH reduced per minute through calculations that used the extinction coefficient of NADPH (6.2mM−1cm−1). The GPOX was normalized for protein content and expressed as pmol of NADPH/min/g.
GSH and GSSG concentration—The GSH and GSSG concentration assays used in this study have been validated in horses26 and were performed according to instructions from a commercially available assay.p The PBMC and RBC lysates were thawed on ice and precipitated with 5% sulfosalicylic acid.q The PBMC samples were diluted 2-fold or did not require dilution for analysis. The RBC samples were diluted 10- to 100-fold for analysis. Samples were added to a 96-well microtiter plate with a working solution containing 5,5′-dithiobis(2-nitrobenzoic acid)r and glutathione reductase.s Following 5 minutes of incubation at 20°C, NADPH solutiont was added and the plate was read immediately. A microplate readerh recorded absorption of samples every 60 seconds for 5 minutes at 412 nm. The resulting slope was plotted from the change in absorbance at 412 nm/min. Standard curves of GSHu and GSSGv were analyzed, and linear regression of the change in absorbance at 412 nm/min was used to estimate the concentration of GSH and GSSG in test samples. Lower limit of quantitation of the assays was 0.5 ng/mL. Estimated concentration of GSH and GSSG was normalized for protein content and expressed in nanomoles per milligram.
RT-PCR assay—An RT-PCR assay was used to quantitate cytokine gene expression in blood samples. Samples were thawed, and total RNA was extracted by use of a commercial kitw and manufacturer's instructions. Samples were converted to cDNA via RT by use of 1.0 μg of RNA sample and RT master mix,x with incubation at 42°C for 15 minutes and 95°C for 5 minutes as described.27 Equine-specific intron-spanning primer and probe sets were used.28 Cytokines evaluated included TNFα, IL-1, and IL-2. Reaction mixtures composed of 5 μL of cDNA, 6.25 μL of nuclease-free water, 1.25 μL of 20× assay mix for the primer-probe set, and 12.5 μL of master mixw were incubated at 95°C for 10 minutes and underwent 40 cycles in a sequence detection system.y β—glucuronidase was used as the housekeeping gene. Changes in gene expression (ΔΔCT) were calculated by use of the following formula29:
Results were reported as relative cytokine gene expression calculated by use of 2−ΔΔCT, calibrated to samples from day 1 prior to administration of the blank diet for each individual gene.
Statistical analysis—Changes in plasma antioxidant capacity, glutathione, antioxidant enzymes in RBCs and PBMCs, and quantity of cytokine mRNA relative to silibinin dose, were analyzed by use of repeated measures ANOVA with Bonferroni multiple comparisons for post hoc pairwise comparisons. Values of P < 0.05 were considered significant.
Results
All 5 horses completed the study. Each 7-day period of twice-daily feeding of silibinin phospholipid was well tolerated.
ORAC—Plasma ORAC was measured for all time points (Table 1). Plasma samples delayed oxidation of the fluorescent probe to a greater extent than did blank samples. Plasma ORAC increased significantly (P = 0.005) throughout the study period.
Plasma ORAC (mean ± SD [median {range}]) of horses orally administered various doses of silibinin phospholipid.
| Silibinin dose (mg/kg) | Sample day | ORAC* |
|---|---|---|
| 0 | 1 | 22.5 ± 1.8a (22.8 [20.2–24.8]) |
| 0 | 7 | 24.8 ± 1.8a (25.0 [22.2–27.7]) |
| 6.5 | 1 | 24.6 ± 1.6a,b (24.1 [22.9–26.9]) |
| 6.5 | 7 | 26.1 ± 1.6b (26.2 [24.0–27.7]) |
| 13 | 1 | 26.5 ± 0.7b,c (26.3 [25.7–27.3]) |
| 13 | 7 | 25.8 ± 0.8b,c (25.9 [24.8–26.7]) |
| 26 | 1 | 27.0 ± 0.7c (26.9 [24.2–27.3]) |
| 26 | 7 | 26.4 ± 1.3c (26.9 [24.2–27.3]) |
Vitamin E analog equivalents per milliliter of plasma protein.
Within a column, values with different superscript letters are significantly (P < 0.05) different.
NQO1 and GPOX activity
Differences in NQO1 activity of PBMC samples were not detected among horses fed different doses of silibinin phospholipid, (Table 2; P = 0.087). In RBC samples, NQO1 activity decreased significantly (P = 0.017) during the study period. Differences in GPOX activity of PBMC (P = 0.357) and RBC (P = 0.248) samples were not detected among dosage groups.
Mean ± SD (median [range]) values (corrected for sample protein concentration) of endogenous antioxidant enzyme activities and glutothione concentrations in PBMCs and RBCs of horses orally administered various doses of silibinin phospholipid.
| Silibinin dose (mg/kg) | Sample day | Sample type | NQO1 activity (nmol of DCPIP/min/mg) | GPOX activity (pmol of NADPH/min/g) | GSH (nmol/mg) | GSSG (nmol/mg) | GSSG:GSH |
|---|---|---|---|---|---|---|---|
| 0 | 1 | PBMC | 235 ± 211 | 9.5 ± 3.9 | 4.16 ± 2.97 | 3.53 ± 1.60 | 1.24 ± 1.14 |
| (190.7 [77.8–591.5]) | (10.7 [5.4–13.6]) | (3.36 [1.36–8.79]) | (3.65 [1.09–5.38]) | (1.03 [0.36–3.19]) | |||
| 0 | 1 | RBC | 1.26 ± 0.86a | 6.3 ± 4.1 | 3.88 ± 1.34 | 0.85 ± 0.18 | 0.24 ± 0.08 |
| (0.82 [0.46–2.52]) | (5.4 [3.1–12.2]) | (3.90 [2.58–5.81]) | (0.86 [0.56–1.00]) | (0.22 [0.15–0.37]) | |||
| 0 | 7 | PBMC | 534 ± 243 | 16.6 ± 6.9 | 5.50 ± 4.50 | 4.36 ± 2.41 | 1.30 ± 1.11 |
| (552.3 [259.3–871.8]) | (15.7 [8.9–27.2]) | (5.14 [0.75–12.77]) | (4.31 [2.21–8.17]) | (0.93 [0.39–3.17]) | |||
| 0 | 7 | RBC | 0.55 ± 0.37a,b | 11.1 ± 4.6 | 2.74 ± 3.63 | 0.28 ± 0.20 | 0.15 ± 0.06 |
| (0.47 [0.18–0.97]) | (7.2 [7.0–15.4]) | (1.10 [1.02–9.24]) | (0.19 [0.15–0.63]) | (0.16 [0.07–0.23]) | |||
| 6.5 | 1 | PBMC | 12.3 ± 8.2 | 10.2 ± 4.0 | 12.0 ± 5.73 | 3.96 ± 2.55 | 0.37 ± 0.19 |
| (12.6 [3.5–22.4]) | (9.7 [6.3–16.7]) | (13.3 [2.44–16.58]) | (2.77 [1.56–6.86]) | (0.41 [0.15–0.64]) | |||
| 6.5 | 1 | RBC | 0.79 ± 0.50a,b | 11.2 ± 5.8 | 5.53 ± 2.25 | 0.50 ± 0.40 | 0.09 ± 0.08 |
| (0.78 [0.20–1.50]) | (12.1 [5.5–18.9]) | (5.01 [3.48–9.13]) | (0.40 [0.16–1.13]) | (0.07 [0.05–0.23]) | |||
| 6.5 | 7 | PBMC | 101 ± 96 | 8.3 ± 6.5 | 16.9 ± 13.2 | 14.9 ± 18.7 | 11.4 ± 23.9 |
| (53.2 [13.5–254.0]) | (6.4 [1.7–15.4]) | (19.67 [0.13–35.04]) | (7.12 [1.35–47.8]) | (1.36 [0.06–54.17]) | |||
| 6.5 | 7 | RBC | 0.48 ± 0.30a,b | 12.4 ± 7.0 | 5.37 ± 2.26 | 0.78 ± 0.51 | 0.19 ± 0.14 |
| (0.51 [0.07–0.90]) | (13.3 [5.3–23.6]) | (5.56 [1.69–7.75]) | (0.63 [0.34–1.66]) | (0.14 [0.05–0.37]) | |||
| 13 | 1 | PBMC | 163 ± 185 | 12.9 ± 4.9 | 5.39 ± 5.19 | 9.50 ± 10.2 | 5.17 ± 6.69 |
| (56.6 [4.2–404.9]) | (13.0 [20.2–7.4]) | (4.38 [0.18–13.2]) | (4.70 [1.36–47.8]) | (1.07 [0.40–15.9]) | |||
| 13 | 1 | RBC | 0.35 ± 0.15b | 12.0 ± 1.0 | 4.24 ± 1.88 | 0.67 ± 0.27 | 0.16 ± 0.05 |
| (0.29 [0.25–0.61]) | (11.8 [11.2–13.6]) | (4.10 [1.99–6.52]) | (0.58 [0.26–0.94]) | (0.14 [0.10–0.23]) | |||
| 13 | 7 | PBMC | 185 ± 231 | 12.4 ± 3.6 | 10.2 ± 8.50 | 4.54 ± 1.83 | 4.53 ± 8.75 |
| (71.5 [19.9–569.5]) | (11.1 [8.2–17.6]) | (8.19 [0.16–21.1]) | (3.98 [3.27–7.68]) | (0.55 [0.19–20.15]) | |||
| 13 | 7 | RBC | 0.37 ± 0.38b | 10.8 ± 3.2 | 3.52 ± 1.08 | 0.62 ± 0.44 | 0.17 ± 0.05 |
| (0.13 [0.07–0.82]) | (9.9 [7.9–16.3]) | (3.32 [2.01–4.98]) | (0.52 [0.21–1.34]) | (0.17 [0.06–0.27]) | |||
| 26 | 1 | PBMC | 200 ± 144 | 11.9 ± 3.9 | 6.45 ± 5.48 | 9.02 ± 9.11 | 9.53 ± 15.8 |
| (194.1 [30.9–264.1]) | (11.9 [6.3–16.8]) | (7.51 [0.10–12.10]) | (4.01 [3.59–24.8]) | (3.30 [0.30–37.44]) | |||
| 26 | 1 | RBC | 0.67 ± 0.57b | 9.3 ± 2.2 | 4.48 ± 0.84 | 0.81 ± 0.44 | 0.17 ± 0.08 |
| (0.38 [0.17–1.43]) | (10.1 [6.9–11.8]) | (4.99 [3.31–5.31]) | (0.88 [0.15–1.29]) | (0.19 [0.03–0.24]) | |||
| 26 | 7 | PBMC | 572 ± 435 | 8.9 ± 4.6 | 5.75 ± 5.84 | 7.84 ± 4.12 | 7.62 ± 13.4 |
| (368.2 [132.3–1231.6]) | (7.6 [3.9–16.2]) | (3.33 [0.24–15.43]) | (7.70 [3.74–14.54]) | (2.37 [0.24–31.6]) | |||
| 26 | 7 | RBC | 0.32 ± 0.09b | 9.1 ± 4.3 | 4.28 ± 1.25 | 0.55 ± 0.27 | 0.12 ± 0.04 |
| (0.32 [0.23–0.47]) | (6.6 [6.1–16.1]) | (4.24 [2.50–5.94]) | (0.55 [0.20–0.88]) | (0.12 [0.08–0.17]) |
Within a column and sample type, values with different superscript letters are significantly (P < 0.05) different.
GSH and GSSG concentrations
Differences in GSH and GSSG concentration of PBMC and RBC samples were not detected among groups (Table 2). Differences in GSSG:GSH ratio were not detected in PBMC samples (P = 0.453). The GSSG to GSH ratio decreased with silibinin phospholipid dose in RBC samples, but this difference was not significant (P = 0.47).
RT-PCR assay
In 1 horse, the RT-PCR assay housekeeping gene was amplified too late, invalidating the cytokine data. Therefore, this horse was excluded and only 4 horses were included in the final analysis. Differences in TNFα, IL-1, and IL-2 expression were not detected among groups. Relative quantities of gene expression calibrated to the first sampling period, day 1 of the unsupplemented diet week, were determined (Table 3).
Blood cytokine gene expression (mean ± SD [median {range}]) of horses orally administerec various doses of silibinin phospholipid.
| Silibinin dose (mg/kg) | Sample day | TNFα | IL-1 | IL-2 |
|---|---|---|---|---|
| 0 | 1 | Referent | Referent | Referent |
| 0 | 7 | 1.17 ± 0.52 | 1.12 ± 0.21 | 1.73 ± 1.51 |
| (1.15 [0.56–1.84]) | (1.13 [0.87–1.36]) | (1.24 [0.51–3.94]) | ||
| 6.5 | 1 | 0.81 ± 0.36 | 1.06 ± 0.19 | 0.98 ± 0.31 |
| (0.87 [0.33–1.18]) | (1.12 [0.79–1.21]) | (0.94 [0.66–1.37]) | ||
| 6.5 | 7 | 0.89 ± 0.56 | 1.30 ± 0.51 | 0.68 ± 0.30 |
| (0.64 [0.56–1.72]) | (1.23 [0.74–1.97]) | (0.68 [0.36–1.02]) | ||
| 13 | 1 | 1.16 ± 0.49 | 1.49 ± 1.27 | 1.04 ± 0.28 |
| (1.28 [0.50–1.56]) | (0.88 [0.79–3.39]) | (1.09 [0.68–1.29]) | ||
| 13 | 7 | 0.85 ± 0.41 | 1.39 ± 0.20 | 1.41 ± 0.62 |
| (0.98 [0.25–1.18]) | (1.43 [1.12–1.60]) | (1.52 [0.66–1.96]) | ||
| 26 | 1 | 0.75 ± 0.37 | 1.04 ± 0.31 | 1.32 ± 0.20 |
| (0.85 [0.23–1.05]) | (0.99 [0.77–1.40]) | (1.29 [1.14–1.57]) | ||
| 26 | 7 | 1.03 ± 0.64 | 1.21 ± 0.45 | 1.31 ± 0.50 |
| (1.03 [0.26–1.82]) | (1.16 [0.73–1.81]) | (1.15 [0.89–2.04]) |
Gene expression values indicate relative quantity of gene calibrated to day 1 of the unsupplemented diet (0 mg/kg) week.
Discussion
The primary antioxidant effect identified in the healthy horses fed diets supplemented with silibinin in the present study was alteration in plasma ORAC. Temporal effects and those of the carrier diet may also have been sources of alterations in plasma ORAC in this study. Modest increases in plasma ORAC have also been associated with decreases in plasma lipid hydroperoxides in horses following exercise-induced stress.6 In the present study, the GSSG:GSH ratio in RBC samples decreased with increased doses of silibinin phospholipid, although this decrease was not significant. Erythrocyte NQO1 activity decreased with increasing dose, but a significant change in PBMC NQO1 activity was not observed. The GPOX activity was not altered with silibinin administration, although silibinin has been reported to have positive effects on antioxidant capacity in blood cells and plasma, including restoration of antioxidant defenses in pathological disease states. Maintenance of GPOX activity is a primary mechanism of silibinin protection against RBC lipid peroxidation and damage secondary to oxidation.30 Silibinin administration significantly increases RBC GPOX activity in humans undergoing hemodialysis31 and ameliorates decreases in serum GPOX activity that occur secondary to experimental renal ischemia and reperfusion in rats.32 Silibinin is also protective against decreases in GPOX activity and GSH concentration in human RBCs that occur secondary to peroxide exposure in vitro.30 Silibinin restores PBMC GSH concentration in humans with β-thalassemia major, and improves PBMC proliferative responses.9 Silibinin also improves the PBMC GSSG:GSH ratio in vitro following oxidant-induced damage.10 In healthy cats, silibinin increases PBMC GSH concentration but does not alter the GSSG:GSH ratio.33 Although ORAC has not been specifically measured following silibinin administration, protective effects on serum and plasma antioxidant capacity have been detected.11,32 Information regarding silibinin effects on NQO1 activity is not available, although dose-dependent increases in NADPH2:quinone reductase have been reported in mice in vivo.4
In a study34 of rats that were administered silibinin via the intraperitoneal route, increases in GSH concentration were tissue specific and occurred primarily in the liver and intestines. The authors attributed the tissue-specific effects to the basic pharmacokinetics of silibinin, which undergoes predominantly biliary excretion and is maintained in high local concentrations by enterohepatic circulation. It is presumed that the antioxidant protective effects of silibinin are similarly concentrated in the tissues of the liver and intestines. Therefore, it is expected that the changes evident in the blood of horses consuming silibinin would be greater in the liver, which is the target organ in horses with liver disease. Hepatic antioxidant effects of silibinin are well documented.35–38 Less is known regarding the relative antioxidant effect on blood versus hepatic tissues. In experimental carbon tetrachloride-induced hepatitis, hepatic and RBC GSH were both measured and silibinin administration improved GSH concentration in both tissues, but to a greater degree in hepatic tissue.39
Much has been learned from in vitro and in vivo experiments with respect to the peripheral anti-inflammatory effects of silibinin administration. Silibinin reverses increases in serum TNFα, IL-Iβ, and IL-6 expression in rats with sepsis induced by cecal ligation and perforation.11 A decrease in acute lung and brain injury accompanies this anti-inflammatory effect.11 In rats with experimental nonalcoholic fatty liver disease, silibinin decreases plasma TNFα expression concurrent with improvements in liver inflammation and fatty infiltration evident via histologic examination.40 In rats, silibinin significantly reduces serum TNFα and IL-1 expression associated with partial hepatectomy and the resultant inflammatory response.41 Release of TNFα and cytotoxicosis secondary to toxic damage are decreased by silibinin administration in perfused livers and isolated Kupffer cells.42 In canine hepatocytes, silibinin ameliorates the proinflammatory influence of IL-1β, including production of chemotactic cytokines, and reduces hepatocyte damage.13 Silibinin also diminishes the proinflammatory influence of IL-1 in human hepatic stellate cells.43
Gene expression of the inflammatory cytokines TNFα, IL-1, and IL-2 did not change in the present study of healthy horses fed diets supplemented with silibinin phospholipid. In the absence of an active inflammatory stimulus, this was not surprising. However, the effects of silibinin on inflammatory cytokines have been detected in the blood of patients with naturally occurring diseases, especially those diseases in which oxidative injury is prominent. In chronic hepatitis C virus infection, silibinin administration inhibits TNFα production by PBMCs.12 Production of TNFα by blood lymphocytes in humans with end-stage diabetic nephropathy is significantly reduced following silibinin administration.44 Silibinin also reduces inflammatory cytokines and disease severity in humans with non alcoholic fatty liver disease.45–47
Results of the present study provide baseline data on the effects of silibinin in healthy horses. Minor changes were observed in antioxidant capacity, which was consistent with previous observations in healthy cats.33 Because of the pharmacokinetics of silibinin,3 despite low bioavailability, its antioxidant effects will likely be most prominent in the liver and intestinal tract of horses.
ABBREVIATIONS
| AUC | Area under the curve |
| β-GUS | β-glucuronidase |
| DCPIP | 2,6-dichlorophenolindophenol |
| GPOX | Glutathione peroxidase |
| GSH | Reduced glutathione |
| GSSG | Oxidized glutathione |
| IL | Interleukin |
| NADPH | Nicotinamide adenine dinucleotide phosphate |
| NQO1 | Nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase I |
| ORAC | Oxygen radical absorbance capacity |
| PBMC | Peripheral blood mononuclear cells |
| RT | Reverse transcription |
| TNFα | Tumor necrosis factor α |
Equine Senior Horse Feed, Purina Mills, St Louis, Mo.
Siliphos, Indena Pharmaceuticals Inc, Milan, Italy.
Sigma Ficoll F4375, Sigma-Aldrich United States, St Louis, Mo.
Sigma Histopaque 1077, Sigma-Aldrich United States, St Louis, Mo.
PreAnalytix PAXgene blood RNA tube, Qiagen, Valencia, Calif.
Sonic Dismembrator, Fisher Scientific, Pittsburgh, Pa.
23225 - BCA Protein Assay Kit, Thermo Scientific, Waltham, Mass.
Synergy HT, Bio-tek Instruments Inc, Winooski, Vt.
Cell Biolabs Inc, San Diego, Calif.
Cell Biolabs 234502, San Diego, Calif.
AAPH, Cell Biolabs 234503, San Diego, Calif.
Trolox, Cell Biolabs 234504, San Diego, Calif.
Sigma D1878, Sigma-Aldrich United States, St Louis, Mo.
NADH, Sigma N4505, Sigma-Aldrich United States, St Louis, Mo.
DU 800 UV/Vis, Beckman Coulter, Brea, Calif.
Sigma-Aldrich United States, St Louis, Mo.
Sigma S2130, Sigma-Aldrich United States, St Louis, Mo.
DTNB, Sigma D8130, Sigma-Aldrich United States, St Louis, Mo.
Sigma G3664, Sigma-Aldrich United States, St Louis, Mo.
Sigma N6505, Sigma-Aldrich United States, St Louis, Mo.
Sigma G4544, Sigma-Aldrich United States, St Louis, Mo.
Sigma G4376, Sigma-Aldrich United States, St Louis, Mo.
PreAnalytix PAXgene blood RNA extraction kit, Qiagen, Valencia, Calif.
Promega, Madison, Wis.
Applied Biosystems, Foster City, Calif.
References
1. West HJ. Clinical and pathological studies in horses with hepatic disease. Equine Vet J 1996; 28:146–156.
2. Durham AE, Newton JR, Smith KC, et al. Retrospective analysis of historical, clinical, ultrasonographic, serum biochemical and haematological data in prognostic evaluation of equine liver disease. Equine Vet J 2003; 35:542–547.
3. Hackett ES, Twedt DC, Gustafson DL. Milk thistle and its derivative compounds: a review of opportunities for treatment of liver disease. J Vet Intern Med 2013; 27:10–16.
4. Zhao J, Agarwal R. Tissue distribution of silibinin, the major active constituent of silymarin, in mice and its association with enhancement of phase II enzymes: implications in cancer chemoprevention. Carcinogenesis 1999; 20:2101–2108.
5. Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. J Agric Food Chem 2005; 53:1841–1856.
6. Kinnunen S, Hyyppa S, Lehmuskero A, et al. Oxygen radical absorbance capacity (ORAC) and exercise-induced oxidative stress in trotters. Eur J Appl Physiol 2005; 95:550–556.
7. Strauss RG, Snyder EL, Wallace PD, et al. Oxygen-detoxifying enzymes in neutrophils of infants and their mothers. J Lab Clin Med 1980; 95:897–904.
8. Vasiliou V, Ross D, Nebert DW. Update of the NAD(P)H:quinone oxidoreductase (NQO) gene family. Hum Genomics 2006; 2:329–335.
9. Alidoost F, Gharagozloo M, Bagherpour B, et al. Effects of silymarin on the proliferation and glutathione levels of peripheral blood mononuclear cells from beta-thalassemia major patients. Int Immunopharmacol 2006; 6:1305–1310.
10. Kiruthiga PV, Pandian SK, Devi KP. Silymarin protects PBMC against B(a)P induced toxicity by replenishing redox status and modulating glutathione metabolizing enzymes-an in vitro study. Toxicol Appl Pharmacol 2010; 247:116–128.
11. Toklu HZ, Tunali Akbay T, Velioglu-Ogunc A, et al. Silymarin, the antioxidant component of Silybum marianum, prevents sepsis-induced acute lung and brain injury. J Surg Res 2008; 145:214–222.
12. Polyak SJ, Morishima C, Shuhart MC, et al. Inhibition of T-cell inflammatory cytokines, hepatocyte NF-kappaB signaling, and HCV infection by standardized Silymarin. Gastroenterology 2007; 132:1925–1936.
13. Au AY, Hasenwinkel JM, Frondoza CG. Silybin inhibits interleukin-1beta-induced production of pro-inflammatory mediators in canine hepatocyte cultures. J Vet Pharmacol Ther 2011; 34:120–129.
14. Tiegs G, Wolter M, Wendel A. Tumor necrosis factor is a terminal mediator in galactosamine/endotoxin-induced hepatitis in mice. Biochem Pharmacol 1989; 38:627–631.
15. Kang YH, Berthiaume F, Yarmush ML. Long-term stable cultures of rat hepatocytes: an in vitro model to study acute and chronic hepatic inflammation. Tissue Eng 2002; 8:681–693.
16. Fisher NC, Neil DA, Williams A, et al. Serum concentrations and peripheral secretion of the beta chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1alpha in alcoholic liver disease. Gut 1999; 45:416–420.
17. Daniluk J, Szuster-Ciesielska A, Drabko J, et al. Serum cytokine levels in alcohol-related liver cirrhosis. Alcohol 2001; 23:29–34.
18. Zi X, Mukhtar H, Agarwal R. Novel cancer chemopreventive effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNF alpha. Biochem Biophys Res Commun 1997; 239:334–339.
19. Schümann J, Wolf D, Pahl A, et al. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am J Pathol 2000; 157:1671–1683.
20. Hackett ES, Mama KR, Twedt DC, et al. Pharmacokinetics and safety of silibinin in horses. Am J Vet Res 2013; 74:1327–1332.
21. Ungvari Z, Sosnowska D, Podlutsky A, et al. Free radical production, antioxidant capacity, and oxidative stress response signatures in fibroblasts from Lewis dwarf rats: effects of life span-extending peripubertal GH treatment. J Gerontol A Biol Sci Med Sci 2011; 66:501–510.
22. Ou B, Hampsch-Woodill M, Prior RL. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem 2001; 49:4619–4626.
23. Ninfali P, Aluigi G. Variability of oxygen radical absorbance capacity (ORAC) in different animal species. Free Radic Res 1998; 29:399–408.
24. Gustafson DL, Siegel D, Rastatter JC, et al. Kinetics of NAD(P)H: quinone oxidoreductase I (NQO1) inhibition by mitomycin C in vitro and in vivo. J Pharmacol Exp Ther 2003; 305:1079–1086.
25. Gustafson DL, Swanson JD, Pritsos CA. Modulation of glutathione and glutathione dependent antioxidant enzymes in mouse heart following doxorubicin therapy. Free Radic Res Commun 1993; 19:111–120.
26. Janiak M, Suska M, Dudzinska W, et al. Blood glutathione status and activity of glutathione-metabolizing antioxidant enzymes in erythrocytes of young trotters in basic training. J Anim Physiol Anim Nutr (Berl) 2010; 94:137–145.
27. Adams AA, Katepalli MP, Kohler K, et al. Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses. Vet Immunol Immunopathol 2009; 127:286–294.
28. Breathnach CC, Sturgill-Wright T, Stiltner JL, et al. Foals are interferon gamma-deficient at birth. Vet Immunol Immunopathol 2006; 112:199–209.
29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001; 25:402–408.
30. Kiruthiga PV, Shafreen RB, Pandian SK, et al. Silymarin protection against major reactive oxygen species released by environmental toxins: exogenous H2O2 exposure in erythrocytes. Basic Clin Pharmacol Toxicol 2007; 100:414–419.
31. Roozbeh J, Shahriyari B, Akmali M, et al. Comparative effects of silymarin and vitamin E supplementation on oxidative stress markers, and hemoglobin levels among patients on hemodialysis. Ren Fail 2011; 33:118–123.
32. Turgut F, Bayrak O, Catal F, et al. Antioxidant and protective effects of silymarin on ischemia and reperfusion injury in the kidney tissues of rats. Int Urol Nephrol 2008; 40:453–460.
33. Webb CB, McCord KW, Twedt DC. Assessment of oxidative stress in leukocytes and granulocyte function following oral administration of a silibinin-phosphatidylcholine complex in cats. Am J Vet Res 2009; 70:57–62.
34. Valenzuela A, Aspillaga M, Vial S, et al. Selectivity of silymarin on the increase of the glutathione content in different tissues of the rat. Planta Med 1989; 55:420–422.
35. Das SK, Vasudevan DM. Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanolinduced oxidative stress in liver. Indian J Biochem Biophys 2006; 43:306–311.
36. Pradeep K, Mohan CV, Gobianand K, et al. Silymarin modulates the oxidant-antioxidant imbalance during diethylnitrosamine induced oxidative stress in rats. Eur J Pharmacol 2007; 560:110–116.
37. Campos R, Garrido A, Guerra R, et al. Silybin dihemisuccinate protects against glutathione depletion and lipid peroxidation induced by acetaminophen on rat liver. Planta Med 1989; 55:417–419.
38. Yao J, Zhi M, Minhu C. Effect of silybin on high-fat-induced fatty liver in rats. Braz J Biol Med Res 2011; 44:652–659.
39. Fahmy SR, Hamdi SA. Curative effect of the Egyptian marine Erugosquilla massavensis extract on carbon tetrachloride-induced oxidative stress in rat liver and erythrocytes. Eur Rev Med Pharmacol Sci 2011; 15:303–312.
40. Haddad Y, Vallerand D, Brault A, et al. Antioxidant and hepatoprotective effects of silibinin in a rat model of nonalcoholic steatohepatitis. Evid Based Complement Alternat Med 2011;2011: nep164.
41. Horváth ME, González-Cabello R, Blázovics A, et al. Effect of silibinin and vitamin E on restoration of cellular immune response after partial hepatectomy. J Ethnopharmacol 2001; 77:227–232.
42. Al-Anati L, Essid E, Reinehr R, et al. Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells. Mol Nutr Food Res 2009; 53:460–466.
43. Trappoliere M, Caligiuri A, Schmid M, et al. Silybin, a component of sylimarin, exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells. J Hepatol 2009; 50:1102–1111.
44. Dietzmann J, Thiel U, Ansorge S, et al. Thiol-inducing and immunoregulatory effects of flavonoids in peripheral blood mononuclear cells from patients with end-stage diabetic nephropathy. Free Radic Biol Med 2002; 33:1347–1354.
45. Loguercio C, Federico A, Trappoliere M, et al. The effect of a silybin-vitamin E-phospholipid complex on nonalcoholic fatty liver disease: a pilot study. Dig Dis Sci 2007; 52:2387–2395.
46. Federico A, Trappoliere M, Tuccillo C, et al. A new silybin-vitamin E-phospholipid complex improves insulin resistance and liver damage in patients with non-alcoholic fatty liver disease: preliminary observations. Gut 2006; 55:901–902.
47. Federico A, Niosi M, Vecchio Blanco CD, et al. Emerging drugs for non-alcoholic fatty liver disease. Expert Opin Emerg Drugs 2008; 13:145–158.
