| Renal ischemia reperfusion frequently prone to ischemia-reperfusion injury(IRI). Renal ischemia reperfusion injury is a common clinical phenomenon of renal injury. Studies have confirmed that curcumin can reduce the renal IRI. It shows the potential value of clinical application. Recent studies have also found that ischemic postconditioning(IPO) had a significant protective effect of renal IRI, the mechanism may be related to reduc apoptosis or oxidative stress. Currently, using curcumin and ischemic postconditioning in combination to reduce renal IRI have not been reported. In this study, we built a rat renal IRI model, with curcumin preconditioning, IPO, curcumin preconditioning combined with IPO treatment. The protective effect of curcumin and IPO on IRI will be observed, then explore the possible protective mechanisms of apoptosis in the curcumin and IPO treatment on rats with acute renal IRI. The study were divided into two parts:(1) Protective effect of curcumin preconditioning, ischemic postconditioning, curcumin preconditioning combined with ischemic postconditioning on renal ischemia reperfusion injury in rats;(2) Effect of curcumin preconditioning, ischemic postconditioning, curcumin preconditioning combined with ischemic postconditioning on rats renal ischemia reperfusion apoptosis and Bcl-2, Bax and Fas protein expression.Part1.Protective effect of curcumin preconditioning, ischemic postconditioning and curcumin preconditioning combined with ischemic postconditioning on renal ischemia reperfusion injury in ratsObjective:To establish a model of renal ischemia reperfusion and investigate the effect of curcumin preconditioning, ischemic postconditioning and curcumin preconditioning combined with ischemic postconditioning on the renal IRI of rats.Methods:Sixty SD rats were randomly divided into five groups, namely sham-operated group(Group Sham), renal ischemia reperfusion model group(Group IR), curcumin preconditioning group(Group CUR), ischemia postconditioning group(Group IPO) and curcumin preconditioning combined with ischemic postconditioning group(Group CUR+IPO), there were12rats in each group. Group Sham:Removed the right kidney and the left one was exposed but its pedicles was not clamped.1ml of0.1%dimethyl sulfoxide(DMSO) was injected into the peritoneal cavity2h before the surgery. Group IR:Removed the right kidney and exposed the left one. The left kidney was treated for45min ischemia and reperfused for24h.1ml of0.1%DMSO injected into the peritoneal cavity2h before the surgery. Group CUR: Removed the right kidney and exposed the left one. The left kidney was treated for45min ischemia and reperfused for24h, curcumin were given2h before the surgery, according to the dose that100mg/kg dissolved in1ml of0.1%DMSO, injected into the peritoneal cavity. Group IPO:Removed right kidney and exposed the left one. The left kidney was treated for45min ischemia, reperfused for10s and added another10s ischemia for six cycles, and then reperfused for24h.1ml of0.1%DMSO was injected into the peritoneal cavity2h before the surgery. Group CUR+IPO:Removed right kidney and exposed the left one. The left kidney was treated for45min ischemia, reperfused for10s and added another10s ischemia for six cycles, and then reperfused for24h. Curcumin were given2h before the surgery, according to the dose that100mg/kg dissolved in1ml of0.1%DMSO, injected into the peritoneal cavity. After reperfused for24h, opened the original incision, the inferior vena venous blood was extracted and the left kidney was removed. Determin the blood creatinine(Cr) and urea nitrogen(BUN). Kidney was longitudinal cuted, half of the renal tissue fixed in4%paraformaldehyde for24h, paraffin-embedded sections, hematoxylin eosin(HE) staining observe the changes in renal structure. Another part of the renal tissue was stored in liquid nitrogen, prepared homogenate, and Superoxide dismutase(SOD) activity, Malondialdehyde (MDA) value were measured.Results:1. Cr and BUN:Compared to Group Sham, the values of Cr, BUN in other groups were significantly high (P<0.05). Compared to the Group IR, the Group CUR, Group IPO and Group CUR+IPO Cr, BUN values significantly decreased at reperfusion for24h (P<0.05). Compared to Group CUR and Group IPO, the Cr value of Group CUR+IPO significantly low (P<0.05).2. The change of SOD activity and MDA value in renal tissue:Compared to Group Sham, Group IR SOD activity decreased, while the value of MDA increased,the differences were statistically significant (P<0.05). Compared to Group IR, Group CUR, Group IPO and Group CUR+IPO had higher SOD activity, while the value of MDA decreased, the differences were statistically significant (P<0.05). Compared to Group CUR and Group IPO, SOD activity of Group CUR+IPO was significantly higher (P<0.05),and the differences of MDA value were not statistically significant (P>0.05).3.The change of tubular structural:Observed with optical microscopy and referenced to Paller renal pathology standard to score. Compared to Group Sham, the renal tubular injury of Group IR was obvious. Compared to Group IR, Group CUR and Group IPO significantly reduced tubular injury, and the Group CUR+IPO more significantly reduced.Conclusion:Curcumin preconditioning and ischemic postconditioning can protect the kidney from IRI. Using the both have a synergistic effect. Part2. Effect of curcumin preconditioning, ischemic postconditioning and curcumin preconditioning combined with ischemic postconditioning on rats ischemia reperfusion renal tubular cells apoptosis and Bcl-2, Bax and Fas protein expressionObjective:To establish a model of renal ischemia reperfusion and investigate the effect of curcumin preconditioning, ischemic postconditioning, curcumin preconditioning combined ischemic postconditioning on rats ischemia reperfusion renal tubular cells apoptosis and Bcl-2, Bax and Fas protein expressionMethods:Sixty SD rats were randomly divided into five groups.The preparation of animal model,group,number of case were same as the first part of this study. Kidney was longitudinal cuted, half of the kidney tissue was fixed in4% paraformaldehyde for24h, paraffin-embedded sections, measured the apoptosis fraction by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), detected the change of Bcl-2, Bax and Fas protein expression by immunohistochemistryResults:1. The results of renal tubular cells apoptosis:TUNEL positive cells obviously increased in Group IR, and with an intensive distribution. The TUNEL positive cells of Group CUR, Group IPO and Group CUR+IPO were less than Group IR, Group CUR+IPO was less than Group CUR and Group IPO, and the difference was significant(P<0.05).2. Bcl-2and Bax protein expression:â‘ Bcl-2protein expression:Compared to Group IR, the Bcl-2protein expression of Group CUR, Group IPO and Group CUR+IPO were increased(P<0.05). Compared to Group CUR, Group CUR+IPO had an increased expression of Bcl-2protein(P<0.05). But compared to Group IPO, Group CUR+IPO had no significant differences on Bcl-2protein expression(P>0.05).â‘¡Bax protein expression:Compared to Group IR, Group CUR and Group IPO were no significant differences on the Bax protein expression(P>0.05), while on Group CUR+IPO the Bax protein expression were decreased(P<0.05). Compared to Group IPO, Group CUR+IPO had a significant decreased expression of Bax protein expression(P<0.05).â‘¢Bcl-2and Bax ratio: Compared to Group IR, Group CUR, Group IPO and Group CUR+IPO had a significantly increased on Bcl-2and Bax ratio(P<0.05); Compared to Group CUR or Group IPO, Group CUR+IPO had a significantly increased on the Bcl-2and Bax ratio(P<0.05).3. Fas protein expression:Compared to Group IR, the Fas protein expression of Group CUR, Group and Group CUR+IPO were decreased (P<0.05). Compared to Group CUR and Group IPO, the Fas protein expression of Group CUR+IPO were decreased(P<0.05). Conclusion:Curcumin preconditioning and ischemic postconditioning can reduce ischemia reperfusion renal tubular cells apoptosis. Using the both agents have a synergistic effect. That may be the reason that they made a protective effect for the renal IRI. Bcl-2protein expression increased, Bax protein expression decreased, Bcl-2and Bax ratio increased, Fas protein expression decreased may be one of the mechanisms. |
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