Garlic antagonizes skeletal muscle ischemia reperfusion injury through regulating inflammation, apoptosis and desmin expression in adult male rats

Abstract

Background: Skeletal muscle injuries with subsequent bleeding is common cause of death on both sports and battle grounds. Application and removal of tourniquet is fast intervention to control hemorrhage resulting ischemia reperfusion (IR) injury. The effect of IR in skeletal muscle is far more severe compared to other body tissues because of the devastating systemic complication. Garlic has beneficial effects in IR of various organs. However, using garlic in IR of skeletal muscle is deficient Goals: To investigate the possible protective effect of garlic in rat model of hind limb IR and its possible mechanisms of action.

Methods: Fifty adult male rats divided into five groups; C: control, IR: ischemia/reperfusion group subjected to 2 hours ischemia followed by 2 hours reperfusion (2/2 hr IR) and three garlic treated groups; G1+IR: 24 hr before I/R, G2+IR: 30 min before IR and G3+IR: immediately before reperfusion. We measured wet to dry weight ratio (W/D) of gastrocnemius muscle, serum creatine kinase (CK), Interleukin 1β (IL-1β), Interleukin-10 (IL-10), gastrocnemius caspase-3 and desmin expression and histopathological damage score.

Results: Garlic treatment caused significant decrease in W/D, serum CK, IL-1β, caspase-3 expression and significant increase in IL-10 as well as desmin expression when compared to IR group. Garlic ameliorated IR-induced histopathological damage and significantly reduced the apoptosis score. Better results obtained with earlier administration before IR.

Conclusion: Garlic protected against IR-induced skeletal muscle damage through reducing inflammation, apoptosis score and elevating desmin expression. We recommend the earlier use of garlic as prophylactic natural medicine in skeletal muscle IR.

Keywords: Ischemia reperfusion (IR); caspase 3; creatine kinase (CK); desmin; garlic; interleukin 1β (IL-1β); interleukin-10 (IL-10); skeletal muscle.

Figure 1

Effect of garlic on wet to dry weight ratio (W/D) of rat gastrocnemeus muscle in different treated groups. C: control group; IR: ischemia reperfusion group; G1+IR: garlic given 24 hours before IR; G2+IR: garlic given 30 minutes before IR; G3+IR: garlic given immediately before reperfusion; One Way ANOVA with Bonferroni posthoc test; *P<0.05, **P<0.01, ***P<0.001, +++P<0.001, ###P<0.001; (*) significance difference of C group vs any other group; (+) IR group vs garlic treated groups; (#) G3+IR vs G1+IR or G2+IR; n=10 in each group.

Figure 2

Effect of garlic on serum creatine kinase (CK) in the studied groups. C: control group; IR: ischemia reperfusion group; G1+IR: garlic given 24 hours before IR; G2+IR: garlic given 30 minutes before reperfusion; G3+IR: garlic given immediately before IR; CK: creatine kinase; IU/L: international unit per liter; One Way ANOVA with Bonferroni posthoc test; ***P<0.001, +++P<0.001, ###P<0.001, &&&P<0.001; (*) significance difference of C group vs any other group; (+) IR group vs garlic treated groups; (#) G3+IR vs G1+IR or G2+IR; (&) P value of G2+IR vs G1+IR; n = 10 in each group.

Figure 3

Effect of garlic on serum interleukin1 beta (IL-1β) (A) and interleukin 10 (IL-10) (B) in the studied groups. C: control group; IR: ischemia reperfusion group; G1+IR: garlic given 24 hours before IR; G2+IR: garlic given 30 minutes before IR; G3+IR: garlic given immediately before reperfusion; IL-1β: Interleukin-1 beta; pg/ml: picogram per milliliter; One Way ANOVA and Bonferroni posthoc test; ***P<0.001, +P<0.05, +++P<0.001, ###P<0.001, &&&P<0.001; (*) significance difference of C group vs any other group; (+) IR group vs garlic treated groups; (#) G3+IR vs G1+IR or G2+IR; (&) G2+IR vs G1+IR; n=10 in each group.

Figure 4

Photomicrographs of caspase-3 immunostaining of rat gastrocnemius muscle (A-E); Apoptosis score (F). (A) control group; (B) IR group; (C) G1+IR group; (D) G2+IR group; (E) G3+IR group; (F) mean area immunostained with caspase 3 ± SD; C: control group; IR: ischemia reperfusion; G1+IR: garlic given 24 hours before IR; G2+IR: garlic given 30 minutes before IR; G3+IR: garlic given immediaty before reperfusion; One Way ANOVA and Bonferroni posthoc test; ***P<0.001, +P<0.05, +++P<0.001, ###P<0.001, &&&P<0.001; (*) significance difference of C group vs any other group; (+) IR group vs garlic treated groups; (#) G3+IR vs G1+IR or G2+IR; (&) G2+IR vs G1+IR; Original magnification 40X; n=4 in each group.

Figure 5

Photomicrographs of desmin immunostaining of rat gastrocnemius muscle. (A-E) longitudinal muscle sections; (F) mean desmin immunostained area in the studied groups; (A) control group showing: strong desmin expression (arrow head) and apparent striations; (B) IR group showing weak or no expression (bifid arrow), loss of lateral alignment of myofibrils and nuclear displacement (thick arrow) in desmin-negative areas; (C-E) rats treated with garlic plus IR, (C) G1+IR showing: increased desmin expression (arrow head), almost normal alignment of skeletal muscle fibers with visible striations (thick arrow) with multiple elongated peripheral nuclei (thin arrow); (D) G2+IR showing increased desmin expression with better striations (asterisk), better alignment of myofibrils (thin arrows), peripherally located nuclei (lightening bolt); (E) G3+IR showing areas weak or no expression (bifid arrow), loss of lateral alignment of myofibrils and myocytes and nuclear displacement (thick arrow) in desmin-negative areas; (F) mean area ± SD; C: control group; IR: ischemia reperfusion group; G1+IR: garlic given 24 hours before IR; G2+IR: garlic given 30 minutes before IR; G3+IR: garlic given immediately before reperfusion; One Way ANOVA and Bonferroni posthoc test; ***P<0.001, +P<0.05, +++P<0.001, ###P<0.001, &&&P<0.001; (*) significance difference of C group vs any other group; (+) IR group vs garlic treated groups; (#) G3+IR vs G1+IR or G2+IR; (&) G2+IR vs G1+IR; original magnification 40X; n=4 in each group.

Figure 6

Photomicrographs of longitudinal sections of rat gastrocnemius muscle (A-E) and total damage score (F). (A) control group; (B) IR group; (C) G1+IR group; (D) G2+IR group; (E) G3+IR group; nuclei (large arrow head), intercellular spaces (asterisk), loss of striation and degenerative areas of muscle fiber (circle), inflammatory cells infiltrations (small arrow head) and hemorrhage between the muscle fibers (arrow); (F) Total damage score ± SD; C: control group; IR: ischemia reperfusion; G1+IR: garlic given 24 hours before IR; G2+IR: garlic given 30 minutes before IR; G3+IR: garlic given immediaty before reperfusion; One Way ANOVA and Bonferroni posthoc test; *P<0.05, ***P<0.001, +P<0.05, +++P<0.001, ###P<0.001, &&P<0.01; (*) significance difference of C group vs any other group; (+) IR group vs garlic treated groups; (#) G3+IR vs G1+IR; (&) G2+IR vs G1+IR; H&E original magnification 40X; n=4 in each group.