| Object:To explore the protective role of Picroside ? on renal ischemia/reperfusion injury and the underlying relationship among autophagy,inflammation and oxidative stress.Methods:40 SD rats were randomly divided into 4 groups(n=10):sham operation group,renal ischemia/reperfusion injury group(RIRI),Picroside ? group(Picroside ?)and Picroside ?+3-MA group.In the sham group,rats underwent a midline laparotomy followed by a right nephrectomy and no other treatment was performed.In the RIRI group,the left kidney vessels were occluded using a clamp for 45min after a right nephrectomy followed by 24h reperfusion.In the Picroside ? group,surgical procedure was the same as that of the RIRI group,and rats were pretreated with Picroside ?(10mg/kg)by intraperitoneal injection at the beginning of reperfusion after ischemia.In the Picroside ?+3-MA group,surgical procedure was the same as that of the RIRI group,and rats were treated with 3-MA(30mg/kg)30min before ischemia followed by Picroside ? treatment(10mg/kg)by intraperitoneal injection at the beginning of reperfusion after ischemia.After 24 hours of reperfusion,blood samples were collected through the inferior vena cava,serum was separated by centrifugation,and serum creatinine(Creatinine,Cr)and urea nitrogen(BUN)were detected by the kit to evaluate renal function.The left kidney was removed immediately after blood collection,and a part of it was stored in 10%formaldehyde,and the other part was immediately stored in liquid nitrogen.Tissues at the junction of the medulla and cortex were cut into the size of 1 mm~3 and were stored in 2.5%glutaraldehyde.H&E staining was performed to assess kidney tissue pathological changes under light microscope.The expression levels of autophagy-related proteins,including LC3?/LC3I and Beclin-1 were detected by Western-blot.RT-PCR was used to detect TNF-?and IL-6 in the kidney tissues.Superoxide dismutase(SOD)and malondialdehyde(MDA)levels were assessed by commercial kit.The number of autophagy lysosomes was evaluated using electron microscope.Results:Compared with the sham group,the serum creatinine and urea nitrogen concentrations in the renal ischemia/reperfusion injury group were significantly increased.The creatinine and urea nitrogen were significantly decreased by Picroside ? pretreatment,and combination of Picroside ? and 3-MA increased the serum creatinine and urea nitrogen concentrations.Simultaneously,no obvious pathological damage was observed in the sham group,and I/R injury induced obvious pathological injury compared with the sham group.While Picroside ? treatment alleviated kidney injury,combination of Picroside ? and 3-MA significantly aggravated tissue damage.In the renal ischemia/reperfusion injury group,the expression levels of LC3?/LC3I and Beclin-1 were higher than those in the sham-operated group,which was further increased by Picroside ? treatment.Combination of Picroside ? and 3-MA significantly down-regulated the expression levels of LC3?/LC3I and Beclin-1.Compared with the sham group,renal ischemia/reperfusion injury caused a significant increase in TNF-?and IL-6,while Picroside ? treatment led to an obvious reduction in TNF-?and IL-6.Combination of Picroside ? and 3-MA resulted in an increase in TNF-?and IL-6.The SOD in the renal ischemia-reperfusion injury group decreased significantly,and the MDA increased significantly when compared with the sham group.Picroside ? treatment increased SOD and increased MDA.While combination of Picroside ? and 3-MA increased SOD and decreased MDA.No obvious autophagy lysosomes were observed in the Sham group,and ischemia/reperfusion could contribute to the information of a few autophagy lysosomes.A great number autophagy lysosomes were found when compared with the I/R group,yet the combination of Picroside ? and 3-MA obviously resulted in an reduction of the number of autophagy lysosomes.Conclusion:Picroside ? could effectively alleviate renal ischemia/reperfusion injury possibly through inhibiting inflammation and oxidative stress by activating autophagy. |
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