Effects Of Pretreatment With Shikonin On ICAM-1,HMGB1 Expression And Apoptosis After Limb Ischemia-reperfusion In Rats

OBJECTIVE: To study the effect of pretreatment with shikonin on the expression of inflammatory mediators and macrophages in rats with limb ischemia-reperfusion injury,and to explore the protective effect of shikonin on skeletal muscle ischemia-reperfusion injury and its mechanism,And to provide a theoretical basis for the new treatment regimen for ischemia-reperfusion injury.Methods: Forty healthy male SD rats were randomly divided into 4 groups(n = 10).Blank group(A),7 days before surgery with intravenous infusion of saline,experimental animals do not do any other treatment.sham experimental group(B): 7 days before surgery with intravenous infusion of saline,separation of femoral artery,not use the arterialclamp to clipping the artery.Control group(C): 7 days before surgery with intravenous infusion of saline,separation of the femoral artery,arterial clip with 4hours of femoral artery,loose arteryclamp blood perfusion.Experimental group(D):7 days before surgery with intravenous infusion of shikonin(10g / kg),experimental separation of the femoral artery,arterial clip with 4 hours of femoral artery,loose arteryclamp blood perfusion.After regular feeding,four groups of preoperative fasting for 6 hours.20% Uralan 0.07 ml / 10 g intraperitoneal injection of anesthesia.In the right incision of the lower abdomen,separation of femoral artery,use the arterialclamp to clipping the artery and blood perfusion in these groups.The model was reconstructed for 24 hours.IL-1,IL-6,TNF-?,HMGB1 and ICAM-1 were measured at 0min-24 H after tailing.The expression of Bcl-2-m RNA and Bax-m RNA in the gastrocnemius muscle were measured by immunohistochemistry.The morphological changes of the gastrocnemius muscle were observed by HE staining.Results: 1.inflammatory mediators Results:The levels of inflammatory mediators in the A group and the B group remained normal and stable.The levels of HMGB1,ICAM-1 and TNF-?,IL-1 and IL-6 in the control group were higher than those in the other groups(P <0.05),and the level of inflammatory cytokines(P <0.05).The levels of serum HMGB1,ICAM-1 and TNF-?,IL-6 and IL-1 in the experimental group were significantly lower than those in the control group(P <0.05).2.RT-PCR results:The expression of Bcl-2 gene in skeletal muscle of group A and group B was significantly higher than that of Bax,and the ratio of Bcl-2 / Bax was decreased,(P> 0.05 for group A and group B)Bax gene expression was further increased,and the ratio of Bcl-2 / Bax was further decreased.(C group and A group,C group and B group,P <0.01).After pretreatment with shikonin,the expression of Bcl-2 gene was higher than that of C group,the expression of Bax gene was decreased and the ratio of Bcl-2 / Bax was higher than C group(P <0.05)3.HE stained light results: A: Skeletal muscle fiber arranged neatly,muscle cells showed no significant degeneration,edema,rupture;interstitial surrounding tissue without significant red blood cell exudation and inflammatory cell infiltration.B: Skeletal muscle fiber arranged neatly,muscle cells showed no significant degeneration,edema,rupture;interstitial surrounding tissue without obvious red blood cell exudation and inflammatory cell infiltration.C: Skeletal muscle fiber edema degeneration,rupture or even dissolution,severe damage to muscle cells,no obvious muscle cell structure,a large number of white blood cells and red blood cell infiltration can be seen between the rupture of dissolved muscle fibers;D: skeletal muscle fibers arranged less regular,edema can still be seen degeneration,but no dissolution of the fracture,muscle swelling was obvious,infiltration of inflammatory cells in muscle fibers was not obvious,but can still be part of the accumulation of vascular damage in the region.Conclusion: 1.After pretreatment with shikonin can inhibit inflammatory reaction by down-regulating the expression of HMGB1 and ICAM-1,and reduce ischemia-reperfusion injury of skeletal muscle of limbs.2.After pretreatment with shikonin can reduce the occurrence of apoptosis by increasing the expression of anti-apoptosis gene Bcl-2 and inhibiting the expression of apoptosis gene Bax,and reduce the ischemic reperfusion injury of skeletal muscle in limbs.