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Abstract

Ischemia–reperfusion is defined as cellular damage after the reperfusion of ischemic tissue, and it is likely to occur in relation to various diseases and surgical procedures. The purpose of this study was to evaluate the capability of atorvastatin to prevent oxidative damage and modulate the release of proinflammatory cytokines in rat hindlimb during ischemia–reperfusion injury. The animals were divided into 4 groups (ischemia–reperfusion + vehicle, ischemia–reperfusion + atorvastatin, sham, and healthy controls) with 15 rats per group. The animals were exposed to ischemia for 6 h, followed by 24 h, 7 days, and 14 days of reperfusion. Atorvastatin was administered by gavage 14 days before ischemia–reperfusion induction. We then measured the serum concentrations and mRNA transcript levels of TNF-α, IL-1β, IL-6, IL-10, SOD2, and CAT. Hematoxylin and eosin stain were performed for histological analyses. Animals subjected to ischemia–reperfusion showed increased serum and transcript levels of TNF-α, IL-1β, IL-6, and IL-10 expressions with a concurrent increase in mRNA transcripts levels compared with sham and healthy controls. Groups treated with atorvastatin showed a significant CAT increase in the first 24 h, but CAT levels decreased at 7 and 14 days. SOD2 enzyme increased in serum without significant changes in mRNA expression. Histological analysis showed inflammatory infiltrate, microhemorrhages, and distortion of the tissue architecture in the first 7 days. At 14 days, the tissue showed loss and damage to myocytes. However, animals treated with atorvastatin showed few histological changes and a decrease in inflammatory cytokines. No significant changes in NO², NO³, or 8-OHdG were observed. Atorvastatin showed a protective effect on the inflammation and tissue damage induced by ischemia–reperfusion in the hindlimb. The antioxidant effect of atorvastatin in the hindlimb is already unclear, and further research is needed to elucidate the molecular mechanism of this drug in the extremities.

Keywords

atorvastatin inflammation ischemia–reperfusion oxidative stress vascular occlusion

Introduction

The chronic interruption of blood flow in the extremities is likely to occur in surgical procedures and cardiovascular diseases because of peripheral arterial occlusion, which causes an acute interruption of blood flow in the limbs. This phenomenon of tissue damage caused by the interruption and subsequent restoration of blood flow is known as ischemia–reperfusion (I-R).¹ The severity of the damage caused by I-R depends on the tissue, the occlusion period, and the time of reperfusion; this damage persists until resolution or total tissue loss due to necrosis and/or apoptosis.² Antioxidant and anti-inflammatory therapies have recently emerged as promising alternatives against the damage caused by I-R.³ The role of statins in recovery from induced I-R injury suggests an antioxidant and anti-inflammatory effect that can ameliorate hindlimb damage; nevertheless, the molecular mechanism is not well understood and further research is required.⁴ Studies have shown that statins have antioxidant and anti-inflammatory properties.⁵ The objective of the current work was to evaluate the role of atorvastatin on cytokines and oxidative damage markers in a minimally invasive and easily reproducible damage-induced model caused by the interruption of blood flow at different times of reperfusion in rat hindlimbs.

Methods

Animals and ethic statements

In this study, 60 male Wistar Kyoto rats (norvegicus albinus) with weights between 250 and 300 gr and ages of 9 to 12 weeks were obtained from the Animal Division of the University of Guadalajara. Animals received care in accordance with the Mexican Official Standard NOM-062-ZOO-1999. The animals were housed at room temperature (18°C–25°C) in a 12-h dark/light cycle with free access to standard diet and filtered tap water. The project was approved and supervised by the Research Ethics Committee of the Health Sciences Center of University of Guadalajara (CI-4647).

Intervention

All animals were divided randomly into 4 groups (I, II, III, and IV) with 15 rats per group. Each of these groups was subdivided into subgroups of 5 animals, which were sacrificed at 24 h, 7 days, and 14 days of reperfusion. Atorvastatin was dissolved in 1 mL of 0.9% NaCl solution and administered to group I by gavage for 14 consecutive days of damage induction at a dose of 30 mg/kg/day. Animals in group II received the same volume of 0.9% NaCl. Groups III and IV did not receive any pharmacological intervention.

I-R induction

In all procedures, animals were intraperitoneally administered with Zoletil 100 (zolazepam and tiletamine; Virbac), which is a general anesthesia, at 40 mg/kg. Once anesthetized, the femoral artery of the right extremity was exposed by blunt dissection, and the femoral vessels were occluded with a 4-0 nylon suture. Animals were re-anesthetized, and ligation was released in groups I and II after 6 h of ischemia.

Sample preparation

Once reperfusion was completed, blood samples were collected under anesthesia (Zoletil 100, 40 mg/kg), and 5–7 mL of circulating blood was collected from the inferior cava vein and centrifuged at 3500 r/min at 4°C for 15 min to separate the serum. The collected blood was then aliquoted and stored at −80°C. The gastrocnemius muscle was removed completely, and a 100 mg portion was stored in RNAlater solution at −80°C. The remaining sample of the gastrocnemius muscle was kept in 4% paraformaldehyde solution.

Histological analysis

Tissues stored in 4% paraformaldehyde solution were embedded in paraffin. Five-micrometer-thick sections were fixed, hydrated, and stained with hematoxylin and eosin (H&E) to evaluate the tissue architecture, inflammatory infiltrate, and microhemorrhages. The full-frame counting scale was used to measure the damage in skeletal muscle.⁶

Inflammation and oxidative stress biomarkers

To measure the serum concentrations of biomarkers, enzyme-linked immunosorbent assay was performed using the following kits: TNF-α (Peprotech), IL-1β (Peprotech), IL-6 (Peprotech) and IL-10 (R&D Systems), 8-OHdG (Trevigen), SOD2 (Elabscience), and CAT (MyBioSource clarifying). All protocols were conducted in accordance with the manufacturing specifications. The concentrations of NO³ and NO² were determined by a colorimetric kit (Sigma).

A semi-quantitative reverse transcription–polymerase chain reaction (PCR) was performed to determine the expression level of our transcripts. The total RNA was extracted using TRIzol. cDNA synthesis was performed using Superscript III reverse transcriptase kit, and PCR was performed with Phusion Hot Start II High-Fidelity DNA polymerase enzyme. The primer sequences were designed using NIH Gen Data Bank, and the target genes were IL-1β (NM_031512), IL-6 (NM_012589), TNF-α (X66539), IL-10 (NM_012854), SOD2 (NM_017051), and CAT (NM_012520). Densitometric analysis was performed with Image J (NIH). Relative mRNA expression was normalized using β-actin as a housekeeping gene.

Statistical analysis

The results are expressed as mean ± standard deviation (SD). For multiple comparisons between groups, two-way analysis of variance (ANOVA) was performed with Tukey’s post hoc test using GraphPad Prism Software 6.0 for Windows (GraphPad Software, Inc., San Diego, CA). Differences with a P-value less than 0.05 were considered significant.

Results

Effect of atorvastatin on histological changes induced by I-R in skeletal muscle

The histological analysis of the gastrocnemius muscle using H&E showed that groups with I-R presented constant structural abnormalities that are commonly described in ischemia (Figure 1). There were no signs of structural damage, but microhemorrhages were observed between the sham and control groups. Only slight inflammatory infiltrate was observed in the sham group (Figure 1). In groups that received doses of 30 mg/kg atorvastatin, the damage to the tissue architecture and the inflammatory infiltrate decreased within 24 h after reperfusion (Figure 1). At 7 and 14 days, there was a notable decrease in inflammatory infiltrate on treated groups in comparison with untreated groups (Figure 1). Full-frame counting analysis showed a decrease in the percentage of damaged myocytes in groups treated with atorvastatin compared with the untreated groups (P < 0.05; Table 1). In addition, a significant reduction in microhemorrhages and inflammatory infiltrate was observed at 24 h, 7 days, and 14 days in the treated groups (P < 0.05 and P < 0.0001, Table 1).

Figure 1.

Effect of atorvastatin on tissue architecture. H&E staining of the gastrocnemius muscle, a representative picture of the histological sections is shown. (a) Control group, (b) ischemia and 24-h reperfusion group, (c) ischemia and 7-day reperfusion group, (d) ischemia and 14-day reperfusion group, (e) sham group, (f) ischemia and 24-h reperfusion group + atorvastatin, (g) ischemia and 7-day reperfusion group + atorvastatin, and (h) ischemia and 14-day reperfusion group + atorvastatin.

Table 1.

Histological analyses.

Reperfusion times	24 Days		7 Days		14 Days
Groups	I	II	I	II	I	II
Damaged myocyte counting^a	43.66 ± 40**	72.50 ± 70	45.66 ± 41**	95.13 ± 88	56.45 ± 57 **	83.50 ± 81
Inflammatory cell counting^a	477 ± 470**	824 ± 725	701 ± 650 *	824 ± 812	502 ± 499 **	892 ± 800
Erythrocytes infiltration counting^a	98 ± 90 **	198 ± 190	192 ± 89*	203 ± 198	149 ± 130**	238 ± 210

Two-way ANOVA comparison between treated group (I) vs non-treated group (II).

Full-frame counting method scales.

P < 0.05; **P < 0.01.

Effect of atorvastatin on serum concentrations and mRNA expression of TNF-α, IL-6, IL-1β, and IL-10

The serum levels of IL-1β decreased in groups treated with atorvastatin at 24 h and 7 days compared with the untreated groups (P < 0.05). Furthermore, the mRNA expression of IL-1β decreased significantly at the same time (P < 0.0001; Figure 2(a) and (b)). We also found a decrease in TNF-α serum concentrations at 7 and 14 days (P < 0.0001) in the treated groups compared with the untreated groups. Although mRNA expression decreased in groups with atorvastatin treatment, the differences were not significant (Figure 2(c) and (d)). The serum levels of IL-6 decreased significantly in groups treated with atorvastatin compared with the untreated groups (P < 0.0001; Figure 3(a)). IL-6 mRNA expression also decreased significantly (P < 0.0001; Figure 3(b)). Changes in IL-10 concentrations at 24 h were not statistically significant. However, at 7 and 14 days, they increased significantly in atorvastatin-treated groups compared with the untreated groups (P < 0.0001; Figure 3(c) and (d)).

Figure 2.

Effect of atorvastatin on (a) serum levels of IL-1β, (b) relative mRNA expression of IL-1β, (c) serum levels of TNF-α (ng/mL), and (d) the relative mRNA expression of TNF-α. Data between groups were analyzed using two-way ANOVA with post hoc Tukey’s test. The results show significant changes between ischemic groups vs controls and between ischemic groups treated with atorvastatin with respect to the untreated groups (*P < 0.05 and **P < 0.0001).

Figure 3.

Effect of atorvastatin in (a) serum levels of IL-6, (b) mRNA expression of IL-6, (c) serum levels of IL-10, and (d) expression of mRNA messenger of IL-10. Results are presented as mean ± SD; data were analyzed using a two-way ANOVA with post hoc Tukey’s test for comparison between groups I-R vs I-R + ATV (*P < 0.05 and **P < 0.0001).

Effect of atorvastatin on oxidative damage in I-R

Although the concentrations of 8-OHdG ng/mL NO³ and NO² were smaller in groups treated with atorvastatin, they were not significantly different (Table 2).

Table 2.

Effect of atorvastatin on oxidative stress markers.

Reperfusion times	24 h		7 Days		14 Days
Groups	I	II	I	II	I	II
SOD2 (mUI/mL)	692 ± 582*	311 ± 365	478 ± 460*	344 ± 327	720 ± 492**	400 ± 300
SOD2 mRNA	1.41 ± 1.25	1.20 ± 1.1	1.96 ± 1.55	1.36 ± 1.20	1.76 ± 1.14	1.04 ± 1.18
CAT (mUI/mL)	1277 ± 116**	465 ± 472	246 ± 230	230 ± 219	372 ± 279	210 ± 211
CAT mRNA	0.55 ± 0.53*	0.146 ± 0.12	0.34 ± 0.28	0.238 ± 0.212	0.41 ± 0.370	0.37 ± 0.32
NO₃ (ng/mL)	0.186 ± 0.28	0.147 ± 0.12	0.186 ± 0.260	0.30 ± 0.22	0.30 ± 0.20	0.285 ± 0.23
NO₂ (ng/mL)	3.07 ± 4.92	4.81 ± 2.93	3.02 ± 4.33	5.27 ± 2.16	3.73 ± 3.6	4.00 ± 3.89
8-OHdG (ng/mL)	121.42 ± 110	114 ± 121	125 ± 121	135 ± 132	110 ± 56	117 ± 110

Two-way ANOVA comparison between treated group (I) vs non-treated group (II). Serum level of all markers was performed using ELISA or colorimetric-based kits. For mRNA expression, we performed a densitometric assay. All data are presented in mean ± SD.

P < 0.05; **P < 0.01.

Effect of atorvastatin on the antioxidant enzymes in I-R

The results show an increase in the serum concentrations of CAT in groups treated with atorvastatin compared with untreated groups, a difference that is significant at 24 h (P < 0.0001; Table 2). Similarly, the relative mRNA expression of CAT increased significantly in the treated groups at 24 hours (P < 0.05; Table 2). However, there was a significant increase in the serum levels of SOD2 in the treated groups compared with the untreated groups at 24 h and 7 days (P < 0.05) and at 14 days (P < 0.0001; Table 2). The mRNA expression of SOD2 increased in the groups that received atorvastatin, but the differences were not statistically significant (Table 2).

Discussion

This is the first report on the effect of atorvastatin in hindlimb I-R. This work shows the protective effect of atorvastatin at a dose of 30 mg/kg against tissue damage, inflammatory infiltrate, and microhemorrhages. Atorvastatin has already shown this effect in I-R conditions.⁷ We observed a concordance with other reports; therefore, to reduce the damage induced by I-R, the modulation of serum levels of cytokines such as IL-1β, IL-6, and TNF-α is required.⁸ It is noteworthy that atorvastatin can reduce the mRNA expression of these inflammation modulating genes in gastrocnemius muscles subjected to I-R.⁹ Some studies report that the anti-inflammatory properties of atorvastatin are attributed to its effect on IL-10 mRNA expression.¹⁰ Our results show an increase in IL-10 levels due to the effect of atorvastatin during the damage resolution phase at 7 and 14 days. Although SOD₂ mRNA expression did not display any significant changes, our results show that the serum levels of SOD2 increased in skeletal muscle; this finding is possibly due to the mechanism of atorvastatin regulation.¹¹ CAT levels were increased after 24 h in groups treated with atorvastatin, thus suggesting that atorvastatin modulates CAT expression; this effect has already been proved with other drugs in skeletal muscle.¹³ However, the NO², NO³, and 8-OHdG levels do not show significant changes. Our results reaffirm that the antioxidant effect of atorvastatin could be dose-dependent.¹² On the basis of these results, we can suggest that atorvastatin has a protective effect against the inflammation and tissue damage induced by I-R in skeletal muscle. However, atorvastatin did not exhibit antioxidant capacity in our model. Further research is needed to elucidate the action mechanism in hindlimb I-R.

Footnotes

All authors contributed to the conceptualization and design of the study and the acquisition,analysis,and interpretation of data. All authors approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research,authorship and/or publication of this article: This project was funded by the Department of Physiology of University of Guadalajara,with partial support granted by the Sectoral Fund for Research in Health and Social Security (CONACYT-FOSISS 2015-1-261442). The author(s) would like to thank the National Council of Science and Technology (CONACYT) for Doctoral Scholarship No. 575523,which enabled the realization of this project.

ORCID iD

Leonel García-Benavides

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Effect of atorvastatin on oxidative damage and inflammation in experimental hindlimb ischemia–reperfusion model