| Background The ischemic tissue and organ will be some damage after its blood restores supply, this phenomenon is called ischemia reperfusion injury（IRI）.Renal IRI is relatively common pathophysiological process in clinical, is one of the important factors leading to ischemic acute kidney injury（AKI）.Renal IRI will occur in many clinical situations, such as surgery, trauma, anesthesia accident,cardio-pulmonary resuscitation,shock, septicemia and renal transplantation. One of the important causes of acute kidney injury is Renal IRI in clinical, Therefore, prevention and treatment of renal IRI is a urgent problem to be solved in clinical.Hydrogen sulfide（H2S） is the third gaseous molecules after nitric oxide（NO） and carbon monoxide（CO） were found in mammals. Endogenous H2 S can be generated in the body and broad participation in physiological and pathophysiological processes of the body.Studies have shown, H2 S has a protective effect on variety of tissue ischemia and hypoxia injury,such as brain, liver and kidney tissue.When the cells are in starvation, it wrapes the sugar, protein or damaged organelles into vesicles, then it is fused with lysosomes formed autophagy lysosome. It uses the lysosomal enzyme degradation within its own damaged cells and macromolecules into small molecules,it can provide energy to cell repair and maintaing energy homeostasis, this procedure is called autophagy, it is different from another programmed cell death mechanism of apoptosis. Studies have shown that H2 S plays a protive role in the liver and heart IRI process through inhibiting autophagy. The study also found that H2 S in the process of renal IRI kidneys play a protective role,but H2 S is by inhibiting or promoting autophagy to enhance autophagy on renal protective effect is unclear.Objective Explore different the protective effect and mechanism of concentrations of sodium hydrosulfide（,NaHS, H2 S donor） produced H2 S pretreatment on a model of mice renal IRI.Methods1. Establishment a model of mice renal IRI and experimental groupsThe experimental has five groups: sham group, IR group, 28μmolNa HS group, 56μmolNaHS group and 100μmolNaHS group. All animals were anesthetized with pentobarbital sodium（75 mg/kg）intraperitoneally before surgery. Cut the back midline of kidney area, blunt dissection muscle tissue surrounding the kidney and renal fascia with a glass minute, then clip the bilateral renal artery gently with a non-invasive artery clip to lead renal ischemia,we will find the kidney color from bright red to pale. After renal ischemia 45 min, loosen the artery clip gently, renal restore blood flow supply immediately, then the kidney color changed from dark red to bright red gradually, all of the above phenomena indicate that the renal reperfusion success. Suture muscle and skin layer by layer, then disinfect the wound. All animals eating chow and drinking water freely. After surgery over 24 h, all mice were killed, then blood and kidneys was collected.2. Detection of mouse kidney function:Using sarcosine oxidase test mouse plasma creatinine（Scr） content; Using urea enzymatic detection mouse plasma urea nitrogen（Bun） content; Using double antibody sandwich ELISA assay mouse plasma cystatin（Cys-c） content.3. Mouse kidney tissue morphology:The mouse kidney tissue was embedded in paraffin after fixing with 4% paraformaldehyde,then cut into paraffin slices. To observe the changes of renal tissue structure, the slices stained with HE staining.The mouse kidney tissue was embedded in epoxy after fixing with 2.5% glutaraldehyde, After uranyl acetate and lead citrate double staining, use electron microscopy to observe ultrastructural.4. Detection of mouse kidney tissue antioxidant:Assay total superoxide dismutase（SOD） vitality in mice kidney tissue by the WST-1 method.Determination malondialdehyde（MDA） content in mice kidney tissue by the TBA method.5. Detection the expression of anti-apoptotic gene Bcl- 2 and pro-apoptotic gene Bax in mice kidney tissue:Detecting the expression of Bcl- 2 and Bax in mouse kidney tissue by Western- blot.6.Detection the expression of LC3,Beclin-1 and P62 in mouse kidney tissue:Detecting the expression of LC3 and Beclin-1 in mouse kidney tissue by Western- blot. Detecting the expression of P62 in mice kidney tissue by Immunohistochemistry.Results1.H2 S pretreatment can improve the changes in renal function significantly caused by renal IRI in mice : Compared with sham group, the plasma Scr, Bun and Cys-C levels of mice in IR group was significantly increased（P <0.01）; Compared with IR group, the plasma Scr and Bun levels of mice in28μmolNaHS group was no significant decline（P> 0.05）, Cys-C was significantly lower（P <0.05）; the plasma Scr, Bun and Cys-C levels of mice in 56μmolNaHS group and 100μmolNaHS groups significantly lower（P <0.05）.2. H2 S pretreatment can significantly improve the kidney structure lesions caused by renal IRI:Compared with sham group, the kidney structure lesions of mice in IR group was significant, HE staining showed that the tubular cell necrosis in the kidney cortex and medulla; The ultrastructural.of renal has been observed in electron microscopy showed that some of the kidney mitochondrial and ridges of mice in IR group have fused while others have disappeard, nuclear-cytoplasmic swelling, endoplasmic reticulum dilatation and degranulation,and perinuclear expansion; Compared with IR group, the kidney disease of mice in 28μmolNaHS group,56μmolNaHS group and 100μmolNaHS groups were significantly improved.3.H2 S pretreatment can significantly improve the renal scavenging capacity of oxygen free radical and total antioxidant capacity within the organization: Compared with sham group, the kidney tissue SOD vitality of mice in IR group was significantly lower（P <0.05） and MDA content of mice in IR group was significantly increased（P <0.05）; Compared with IR group, the kidney tissue SOD vitality of mice in28μmolNaHS group,56μmolNaHS group and 100μmolNaHS groups were significantly increased（P <0.05）and MDA content of mice in 28μmolNaHS group,56μmolNaHS group and 100μmolNaHS groups were significantly lower（P <0.05）.4.H2 S pretreatment can significantly improve kidney cell apoptosis: Compared with sham group, the expression of Bcl-2 in IR group was significantly lower（P <0.05）,but the expression of Bax and Caspase 3 in IR group was significantly increased（P <0.05）. Compared with IR group, the expression of Bcl-2 in 28μmol Na HS group was significantly increased（P <0.05）, but the expression of Bax and Caspase 3 were no significant decline（P> 0.05）; the expression of Bcl-2 in 56μmolNaHS group and100μmolNaHS groups were significantly increased（P <0.05）, the expression of Bax and Caspase 3 were significantly lower（P <0.05）.5.H2 S pretreatment can significantly improve the level of autophagy in kidney: Compared with sham group, the expression of P62 in IR group was significantly increased（P <0.05）, the expression of LC-3Ⅱ/Ⅰand Beclin1 in IR group were significantly increased（P <0.05）. Compared with IR group, the expression of LC-3Ⅱ/Ⅰand Beclin1 in 28μmol Na HS group were no significant decline（P> 0.05）; the expression of LC-3Ⅱ/Ⅰand Beclin1 in 56μmol Na HS were no significant decline（P> 0.05）, but the expression of Beclin1 in 56μmol NaHS group was significantly increased（P <0.05）;the expression of LC-3Ⅱ/Ⅰand Beclin1 in 100μmol Na HS group were significantly increased（P <0.05）;the expression of P62 in 28μmol NaHS, 56μmol Na HS group and 100μmolNaHS groups were significantly lower（P <0.05）.Conclusion1.The partly protective effects of H2 S on renal IRI by increasing renal antioxidant and anti-apoptotic ability.2. The partly protective effects of H2 S on renal IRI by enhancing autophagy.3. The protective effects of H2 S on renal IRI in 56μmolNaHS and 100μmolNaHS groups more obvious. |
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