Protective Effect Of Recombinant Rat Adrenomedullin Gene On Skeletal Muscle Ischemia-reperfusion Injury

Objective:In this study, we constructed rrADM by pVAX1 the eukaryotic expression vector, then direct injection into gastrocnemius of rat. We evaluated the protective effect of rrADM in skeletal muscle on ischemia-reperfusion model and compared the differences of the protective effect in skeletal muscle ischemia-reperfusion injury between rrADM, edaravone, verapamil, allopurinol and papaverine.Methods:1. Take the total rat adrenal RNA from a SD rat and using RT-PCR synthesizes cDNA of ADM by reverse transcription and amplified by PCR, and then the product was cloned to the PMD-18T vector and sub-cloned into pVAX1 the eukaryotic expression vector to obtain plasmid DNA of rrADM. Extract large number of plasmid DNA by alkaline lysis method and purified the plasmid DNA by using polyethylene glycol precipitation classification method. Calculate the concentration of plasmid DNA.2.18 SD rats,203±18.4g were selected and divided into 3 groups randomly, each of which contained 6 rats:control group, empty vector group and 700μg·kgbw-1rrADM group. The 3 groups was direct injected lml NS, 1ml saline solution of pVAX1 plasmid DNA(700μg·kgbw-1) and 1ml saline solution of rrADM plasmid DNA(700μg·kgbw-1) into left hind limb gastrocnemius muscle of rat, once a week for 4 weeks. Obtained the muscle of injected site after 4 weeks and detected the expression of ADM by immunohistochemistry in each group.3.64 SD rats,203±18.4g were selected and divided into 8 groups randomly, each of which contained 8 rats:control group(C), ischemia-reperfusion model group(IR), empty vector group(Ev), rrADM group, edaravone group(Ed), verapamil group(Vp), allopurinol group(Ap) and papverine group(Pp). Ev group and rrADM group were injected 1ml saline solution of pVAX1 plasmid DNA and rrADM plasmid DNA into gastrocnemius muscle respectively 4 weeks before operation, and other 6 groups were injected same dose of normal saline into gastrocnemius muscle. Injected drugs once a week for 4 weeks, then take the skeletal muscle ischemia-reperfusion experiment after 4 weeks, each group was exposed to 3h ischemia and 3h reperfusion. Ed group, Vp group Ap group and Pp group were injected intraperitoneally with 1ml 0.5mg/kg saline, 1ml 2mg/kg saline solution of Vp, 1ml 50mg/kg saline solution of Ap and 1ml 5mg/kg saline solution of Pp respectively, and other 4 groups were injected the same dose of normal saline. After operation, obtained the gastrocnemius muscle tissue of rats and collected serum, and measured content of creatine kinase(CK) and lactic dehydrogenase(LDH) of serum; measured content of malonaldehyde(MDA) and total superoxide dismutase(T-SOD) of muscle tissue; and have pathological examination on muscle tissue.Results:1. Compared the obtained gene sequences with the ADM sequences in Genebank, the two were completely identical. And the results of digested rrADM XbalⅠand HindⅢwere positive. Purified plasmid DNA:OD260=0.912, OD280=0.498, OD260/OD280= 1.831. The concentration of obtained plasmid DNA was 4.560mg/ml.2. The immunohistochemistry result of injected gastrocnemius muscle tissue shows:control group and empty vector group have low levels of expression of ADM in gastrocnemius muscle tissue, and the result was negative; the expression of ADM in gastrocnemius muscle tissue level was high in 700μg·kgbw-1 rrADM group, positive.3. Compared with the content of CK, LDH of serum and MDA of muscle tissue in IR group, the levels were significantly lower(P＜0.01) in rrADM group, Ed group, Vp group, Ap group and Pp group; compared with T-SOD content of muscle tissue in IR group, the levels were significantly higher(P＜0.01) in rrADM group, Ed group, Vp group, Ap group and Pp group. The indexes between IR group and Ev Group were not statistically significant (P＞0.05).Compared with Ap group, the content of LDH of serum in rrADM group was decreased 18.20%(P＞0.05), and the differences of other indexes among rrADM group, Ed group, Vp group, Ap group and Pp group were not stastically significant(P＞0.05). Pathological examination showed that at the injected site of skeletal muscle was observed have large-scale structural confusion in muscle tissue, boundary ambiguity, more inflammatory cell gathered and exuded and tissue have severe edema. However, slight myofilament fracture or tissue edema was hardly observed in rrADM group, Ed group, Vp group, Ap group and Pp group, and a little inflammatory cell gathered even not seen, structure of muscle tissue was normal and pathological changes better than IR group and Ev group.Conclusions:1. Obtained rat ADM gene and recombined rat ADM successfully, extracted and purified rrADM plasmid DNA in accordance with requirements of drugs used in animal experiments.2. After injected rrADM plasmid DNA into gastrocnemius muscle, the ADM gene in tissue was up-regulated. That showed the construct of plasmid was successful, and the transfect and uptake of plasmid were good, which can be used for the next stage of the experiment.3. rrADM can improve the T-SOD activity of the rat skeletal muscle and lower the content of MDA in muscle tissue and plasma CK and LDH levels, has protective effect on rat skeletal muscle ischemia-reperfusion injury. Edaravone, verapamil, allopurinol and paraverine also have protective on rat skeletal muscle ischemia-reperfusion injury, and the differences of protective effect among the drugs were not significantly.