| Ischemia can lead to the trauma of the tissue and the organ,to recover the blood supply is important to make sure its structure and function.But for the issue lacking blood for a long time,recover its blood supply can lead to even severe trauma,it is called ischemical reperfusion injury.Ischemical reperfusion injury is a common pathology process.Using the tourniquet for a long time,the operation of the injury of blood vessel,acute arterial embolism,and limb replantation can lead to the ischemical reperfusion injury.It doesn't only cause issue injury but also can lead to amputation, and multi-organ injury,such as the nonfunction of the liver,kindney,lung and so on, and can finally jeopardize the patients' life.How to lessen the damage of the ischemical reperfusion injury,cut down the mutilation rate and death rate is a warm spots that be studied.Until recently,there are these mechanisms of the ischemical reperfusion injury:the effect of the oxygen free radical and the NO,calcium overload, the participate of various kinds of cytokines and the stimulating of white cell and endotheliocyte making the microcirculation disturbance,and so on.On the basis of these,the study of the drug of preventing the ischemical reperfusion injury gradually goes deeply.Many studies have found that drugs such as vitamin E,manicol,propofol and EPO can obviously protect the limbs from the ischemical reperfusion injury.And the study of the effect of the traditional Chinese medicine also be valued.The shenfu injection is tritused based on the traditional medicine,a drug that can cure shock,it is made by separating sterile water solution.Its effective ingredient is panaxsaponin and aconite alkaloid and so on.It can resist the stress,antioxygen,strengthen the nonspecific resistance and improve the microcirculation.In this study we established the model of the ischemical reperfusion injury of the bandicoot,evaluate the NO level in the blood,the activity of SOD in gastrocnemius issue,the density of Maleic Dialdehyde and the load of Ca2+;extract the total RNA in gastrocnemius issue,and adopt the RT-PCR semiquantitative analysis to get the transcriptional level of Bcl-2-mRNA and Bax-mRNA;and observe the histomorphology change of the gastrocnemius issue.By observing impact of the shenfu injection on the above index, to probe the mechanism of its protection on the ischemical reperfusion injury.Material and MethodsWe adopted 40 healthy SD rats from the exerimental animal center of Academy of medical science of Zhejiang province.Regardless of male and female,weights 200g to 300g.NPO 12 hours before operation,and drink freely.Inject 3 percentage Pentobarbital(made by distilled water and pentobarbital)30mg/kg to the abdominal. Use the rubber tourniquet to ligature the hind limb and block the blood to make the hind limb ischemia at once and become pale and the temperature decreases.Solution the ligation after 4 hours,retrieve the blood for 4 hours,the hind limb become red again,and has made the model of ischemia and reperfusion again.The test animals were divided into 5 groups randomly,and 8 rats every group.Group A,only anesthetize and inject NS to the vena caudalis;group B0,ischemia for 4 hours but not reperfusion;group B4,ischemia for 4 hours and reperfusion for 4 hours;group C0, ischemia for 4 hours after using shenfu injection,but not reperfusion;group C4, ischemia for 4 hours after using shenfu injection and reperfusion for 4 hours.Inject Shenfu injection(made in ya'an sanjiu medicine company)(10ml/kg)to the vena caudalis 10min before the ischemia of the limb,inject the same dosage NS to the rats of group A,B0 and B4.Dissect the rats from the five groups,get blood prepatration from the IVC,and measure the level of NO.Get the tissue from hind limb gastrocnemius and measure the activity of SOD,density of MDA and Ca2+and semi-quantitative analyse the transcriptional level of Bcl-2-mRNA and Bax-mRNA. Use the HE dyeing light microscope the observe the change of the morphology,and the TEM to observe the apoptosis.Use SPSS 15.0 to analyse the data.All data were demonstrated in(?)±s,and adopted analysis of variance,and p＜0.05 demonstrates the significant statistical significance.Result1.The result of Ca2+charge(Unit:mg/kg WW of org.):There was significant increasing of Ca2+charge in group B0,B4 than in group A,B0(156.99±20.47 mg/kg,211.33±53.48 mg/kg VS 7.18±1.86 mg/kg,156.99±20.47 mg/kg,respectively,P＜0.01,P＜0.05),and there was significant decreasing of Ca2+charge in group C0,C4 than in group B0,B4(30.38±5.86 mg/kg,157.64±19.47 mg/kg VS 156.99±20.47 mg/kg,211.33±53.48 mg/kg,respectively,P＜0.01,P＜0.05).2.The result of SOD(Unit:U/mg org.):There was significant decreasing of SOD in group B0,B4 than in group A,B0 (1.59±0.05 U/mg org.,1.06±0.08 U/mg org.VS 1.88±0.16 U/mg org.,1.59±0.05 U/mg org.,respectively,P＜0.01),and there was significant increasing of SOD in group C0,C4 than in group B0,B4(1.76±0.10 U/mg org.,1.61±0.05 U/mg org.VS 1.59±0.05 U/mg org.,1.06±0.08 U/mg org.,respectively,P＜0.01).3.The result of MDA(Unit:nmol/g org.):There was significant increasing of MDA in group B0,B4 than in group A,B0 (71.02±10.37nmol/g org.,97.31±19.68 nmol/g org.VS 23.33±6.31 nmol/g org., 71.02±10.37 nmol/g org.,respectively,P＜0.01),and there was significant decreasing of MDA in group C4 than in group B4(52.32±8.36 nmol/g org.VS 97.31±19.68 nmol/g org.,P＜0.01).But there was no significant change in group C0 than in group B0(60.56±14.95nmol/g org.VS 71.02±10.37 nmol/g org.,P＞0.05).4.The result of NO(Unit:μmol/l): There was significant increasing of NO in group B0,B4 than in group A,B0 (48.70±10.25μmol/l,108.84±14.51μmol/l VS 21.04±5.47μmol/l,48.70±10.25μmol/l,respectively,P＜0.01),and there was significant decreasing of NO in group C0,C4 than in group B0,B4(26.30±5.49μmol/l,69.82±16.35μmol/l VS 48.70±10.25μmol/l,108.84±14.51μmol/l,respectively,P＜0.01).5.The result of RT-PCR:After limb ischemia,the expression of Bcl-2 and the proportionality of bcl-2/bax began to decrease,but the expression of Bax began to increase(B0 compare with A,P＜0.05);Reperfusion lead to the the expression of Bcl-2,the proportionality of bcl-2/bax decreasing and the expression of Bax increasing fourthly(B4 compare with B0,P＜0.01).Treated with shenfu injection,the expression of Bcl-2 and the proportionality of bel-2/bax began to increase obviously,but the expression of Bax began to decrease in medication administration team(C0 compare with B0,C4 compare with B4,P＜0.05).6.The result of Hematoxyline eosin(HE)stain under light microscope:Before ischemia,muscle fibers 'caliber was uniformity and the array of muscle fibers was to line up in order.After ischemia,muscle fibers were in condition of oedema,their array were out of order,part of muscle fibers were to dissolve,and there were small amounts of leucocytes and red blood cells leak out between muscle fibers.After reperfusion,muscle fibers had hydropic degeneration,fragementation even solution and myocyte were destroyed severely.A few of leucocytes and red blood cells can be seen between muscle fibers.Treated with shenfu injection (groupC0,C4),the oedema of skeletal muscle were to lessen obviously,myofibrosis became to slight and the infiltrate of inflammatory decreased compared with control group.7.Characteristic of muscle fibers under transmission electron microscope:Before ischemia,the array of muscle fibers is in order,the structure of cell nucleus is clear;The structure of mitochondria is clear.After ischemia,the spatium intermusculare widen,the structure of myofilament became disorder,the cell nucleus became small,chromatin became margination,the nucleolus became blur;The mitochondria became swelling and its polarity expanded.After reperfusion,the apoptosis of skeletal muscle cell aggravated furtherly.Treated with shenfu injection (groupC0,C4),the muscle fibers became to line up in order,the structure of nucleus became clear.The apotosis of muscle cell became lessened obviously;The swelling and disorganization of mitochondria became slight compared with control group.ConclusionThis study demonstrates that shenfu injection can protect the rats from ischemical reperfusion injury by lessening the calcium overload,reinforcing the activity of the SOD,getting rid of the the oxygen free radical and up-regulating the express of the bcl-2 and making the express of Bax decline and inhibiting the apoptosis of cells of the somatic muscles.It provides basis for the selecting of medicine to decrease the ischemical reperfusion injury. |
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