| Background:Acute lower limb ischemia reperfusion injury is to point to in a short period of time of acute lower limb ischemia, and when restore the blood flow lead to more serious injury. Common in abdominal aortic aneurysm, acute arterial embolism disease such as surgical treatment for a long time to use the clip with blood hemostasis after recovery of blood flow. Acute lower limb ischemia reperfusion injury often accompanies a body inflammatory reaction and tissue damage, or even damage the heart, lung, kidney and circulatory system.Hydrogen has the weak reducibility, can play a role of antioxidant, can selectively remove hydroxyl free radicals.Because the hydrogen molecule is lesser, can through the cell membrane into the organelles, therefore as ischemia-reperfusion control factor has great advantages, attracted many scholars research of hydrogen on ischemia-reperfusion protection. This topic for lower limb in rats with acute ischemia-reperfusion injury model, intends to explore hydrogen to lower limbs of rats acute ischemia-reperfusion injury of protection, and discusses its action mechanism.As a result of the hydrogen gas form a lot of problems exist in the process of application, so we will use the hydrogen saline were studied. Objective:Through the establishment of lower limb in rats with acute ischemia reperfusion model. Study the protection of hydrogen molecules to the lower limbs of acute ischemia-reperfusion in rats and its mechanism of action. Provide theoretical basis for the clinical application of hydrogen molecules to ease the treatment of acute lower limb ischemia-reperfusion injury. Method1. Acute lower limb ischemia reperfusion model: free femoral artery and femoral vein and femoral nerve, free femoral artery about 10 mm, use Bulldog noninvasive blood vessel blocking clamp clip femoral artery, detect blood pressure in the distal femoral artery, when blood pressure is zero mark ischemia success; remove the artery clamp when arrived at the scheduled time point(this study is ischemia 4 hours), blood pressure rise after restore the lower limb blood marks successful reperfusion.2. In long-term drug experiments, after the success of rats acute lower limb ischemia reperfusion, we detect weight of rats on day 1, 3, 5, 7, 9, and adopt posture reflex experiment and body asymmetry methods as behavior evaluation index, to syudy the influence of hydrogen saline on rats motor function recovery after ischemia-reperfusion injury.3. In short-term dosing experiments, 15 hours after lower limb ischemia reperfusion model, kill rats and extract lower limb muscle and liver, lung, kidney tissue, then formaldehyde fixed, paraffin embedding, frozen section biopsy, with HE staining observe tissue morphological structure changes and inflammatory infiltrates.4. Tissue damage detection:(1) tissue section dyed with HE staining, and watch morphological structure changes and inflammatory infiltrates.(2) the use of automatic biochemical analyzer test myoglobin(MB), lipoprotein complex(LPC), lipid peroxide(LPO), and other tissue necrosis breakdown products.(3) using colorimetry to detect in skeletal muscle tissue plasma lactate dehydrogenase(LDH), creatine kinase(CK) levels.5. Inflammatory degree detection: using ELISA method to determine each group, IL- 6, IL- 1 in serum TNF alpha, concentration of HMGB1.6. Oxidation degree detection: colorimetric method is used to detect tissue and mitochondria, the content of MDA, GSH and GSSG in observation group and mitochondrial oxidative reaction degree.7. Mitochondrial structure and function detection:(1) the differential centrifugation separation of muscle cell mitochondria and cytoplasm.(2) the tem observation of skeletal muscle cells mitochondrial morphology change.(3)using spectrophotometry to detect the change of the mitochondrial membrane potential and mitochondrial ATP, Clark oxygen electrode method to detect mitochondrial respiratory function, observe mitochondrial damage.8. Apoptosis detection:(1) the skeletal muscle tissue apoptosis with TUNEL staining method measurement.(2) using western blot method detecting cytochrome C in the organization, Bax, Pro-caspase9 and caspase3 apoptosis related proteins. Results:1. The effect of hydrogen saline to tissue injury after acute lower limb ischemia reperfusion.(1) According to histomorphology, the results of skeletal muscle or remote organs of liver, lung, kidney, hydrogen saline treatment group than control group in the tissue damage caused by lower limb ischemia reperfusion significantly reduce, visible organization form relatively intact, necrotic tissue cell apoptosis, and inflammatory cells infiltrating significantly reduce, etc.(2) The hydrogen saline can significantly reduce the lower limb ischemia reperfusion of skeletal muscle and in the organization apart myoglobin(MB), lipoprotein complex(LPC), lipid peroxide(LPO) and other tissue necrosis of the decomposition product.(3) We also detected the experimental group compared with control group rats serum lactate dehydrogenase(LDH), creatine kinase(CK).2. The effect of hydrogen saline to inflammatory reaction after acute lower limb ischemia reperfusion.Normal saline group rats serum inflammatory factors, IL- 6, IL- 1 TNF alpha, HMGB1 levels increased significantly in the control group, saline group and hydrogen in the rat serum inflammatory cytokines, IL- 6, IL- 1 TNF alpha, HMGB1 levels significantly decreased in the normal saline group.3. The effect of hydrogen saline to oxidative stress after acute lower limb ischemia reperfusion.Lower limb ischemia reperfusion rat skeletal muscle and liver, lung, and kidney tissue MDA and GSSG level increased significantly, and the content of GSH decreased obviously, indicating ischemia reperfusion of skeletal muscle and liver, lung, and kidney tissue of the oxidative stress significantly enhanced, while hydrogen saline treatment can significantly reduce the levels of MDA and GSSG, and improve the content of GSH.4. The effect of hydrogen saline to recovery of motion function after acute lower limb ischemia reperfusion.After ischemia reperfusion rat posture reflex function is impaired, in 1 day, 3 day, 5 days statistically significant difference, and after 7 days, posture reflex function recovered, and hydrogen saline treatment makes posture reflex of rats impaired ease, with 3 and 5 days have significant difference; After ischemia-reperfusion rats body symmetry, in 1 day, 3 day statistically significant difference, after 5 days, body symmetry recovered, and hydrogen saline treatment makes the rat body symmetry damage loss, there are significant differences in 3 days.5. The effect of hydrogen saline tomuscle mitochondria after acute lower limb ischemia reperfusion.(1) Transmission electron microscope you can see that the lower limb ischemia reperfusion rat skeletal muscle cells mitochondrial shape in size shape is irregular, mitochondria swelling, crest disorders is not clear, mitochondrial matrix condensation; And hydrogen saline treatment group rat ischemia-reperfusion rats skeletal muscle cells form uniform size, mitochondrial cristae mitochondria and mitochondrial matrix structure clear rules and less swelling.(2) After lower limb ischemia reperfusion in rats, mitochondrial membrane potential were compared with the control group significantly reduced, and the hydrogen saline treatment group of mitochondrial membrane potential is higher than the control group. This suggests that hydrogen saline treatment can lower limbs by ischemia-reperfusion help maintain their mitochondria membrane potential energy, maintain mitochondrial osmotic balance and protect the mitochondria structure from damage.(3) By detecting different group of lower limb skeletal muscle cells of rats, the content of MDA, GSH and GSSG in mitochondria found lower limb ischemia reperfusion group rats the content of MDA, GSH and GSSG increased compared with control group, saline group and hydrogen than normal saline groups the content of MDA, GSH and GSSG decreased obviously.(4) The lower limb ischemia reperfusion rats can strengthen the consumption of skeletal muscle cells in the mitochondrial ATP, increasing the extent of the oxidation reaction of mitochondria, and hydrogen saline group of rats in the mitochondrial ATP consumption significantly lower than the normal saline group. Hydrogen is consistent with the physiological saline group in the mitochondria of rats p/o ratio significantly higher than the normal saline group.6. The effect of hydrogen saline to skeletal muscle tissue apoptosis after acute lower limb ischemia reperfusion.(1) TUNEL staining results show that the control apoptosis cells is less, and were compared with control group after ischemia-reperfusion in rats skeletal muscle tissue apoptosis, and apoptosis hydrogen saline treatment group compared with control group decreased obviously.(2) In the control group, the expression of Bax mainly in the cytoplasm, and the expression of Bax in control group significantly enhanced in the mitochondria, and expressed in the cytoplasm drops, explains Bax when lower limb ischemia-reperfusion injury by cytoplasm as mitochondria transfer, and result in the expression of Bax after hydrogen saline intervention compared with control group in the cytoplasm increases, relative expressed in mitochondria in the control group. The expression of cytochrome C and Bax show the opposite trend.(3) After ischemia-reperfusion control Pro-caspase9 and caspase3 expression in skeletal muscle cells were significantly higher than the control group, the statistical significant difference; And hydrogen saline after processing, Pro-caspase9 and caspase3 expression in skeletal muscle cells was significantly lower. Conclusion:In this paper, with acute lower limb ischemia reperfusion injury model in rats, and hydrogen saline treatment during reperfusion, in rat skeletal muscle injury after ischemia reperfusion, inflammatory reaction, apoptosis, oxidative stress, mitochondrial function, such as testing and analysis, get the following conclusion:1. Hydrogen saline can significantly reduce lower limb skeletal muscle and the remote organ damage induced by ischemia reperfusion.2. Hydrogen saline can effectively inhibit oxidative stress caused lower limb ischemia reperfusion.3. Hydrogen saline can effectively reduce inflammatory reaction induced by ischemia reperfusion of skeletal muscle of lower limbs.4. Hydrogen saline can significantly improve lower limb motor function recovery in rats after ischemia reperfusion.5.Hydrogen saline can significantly reduce ischemia reperfusion of skeletal muscle mitochondrial damage.(1) hydrogen saline solution can protect the ischemia reperfusion, the structure of mitochondria.(2) hydrogen saline can inhibit skeletal muscle cells mitochondrial ATP consumption, breath control and p/o ratio reduced to protect mitochondrial function.6.Hydrogen saline can inhibit lower limb ischemia reperfusion of skeletal muscle cell apoptosis.(1) hydrogen saline can adjust cytochrome C in skeletal muscle cells and the expression of Bax apoptosis proteins.(2) hydrogen saline control Pro- caspase9 and caspase3 expression in skeletal muscle cells.Taken together, our study shows that the hydrogen tail intravenous saline can significantly inhibited rat acute lower limb ischemia reperfusion cell mitochondrial structure and function caused by injury, and thus enhanced the antioxidant capacity of mitochondria, mitochondrial oxidative stress, reduce the mitochondrial oxidative damage, in addition, the hydrogen saline makes mitochondrial membrane potential energy to maintain, regulation of Bax and cytochrome C in mitochondria and distributed in the cytoplasm, and suppress the activation of Pro- caspase9, inhibit the expression of apoptosis caspase3 downstream molecules, thus inhibiting apoptosis. Skeletal muscle can be reduced by hydrogen tail intravenous saline and remote organs of liver, lung, and kidney oxidative stress, increase the antioxidant ability of organization, and can inhibit skeletal muscle and remote organs of inflammatory cells in the liver, lung, and kidney gathered and release of inflammatory factors, significantly reduce the degree of inflammation tissue and thus effectively protect the lower limb ischemia reperfusion of skeletal muscle and organ away the dysfunction of liver, lung, kidney, lent itself to the restoration of motor function of rats and significantly improve the rat body state. For application of hydrogen saline in auxiliary treatment of lower limb ischemia reperfusion injury to provide theoretical support. |
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