| Objective: To establish a model of renal injury induced by cerebral ischemia-reperfusion in rats by Zea-longa surgery,to evaluate the role of SIK2 in regulating autophagy on acute kidney injury,and to reveal molecules of acute kidney injury during cerebral ischemia-reperfusion.The mechanism provides theoretical and experimental basis for the effective prevention and treatment of acute kidney injury in patients with stroke.Methods: Forty SPF male SD rats were randomly selected and divided into sham operation group(n= 10)and experimental group(n = 30).The experimental group was divided into 3 groups,namely 3h ischemia,6h reperfusion group,12 h group and 24 h group.Rat cerebral ischemia-reperfusion models were established.In the sham operation group,only arterial blood vessels were isolated without plugging,and the rest of the operations were the same as in the experimental group.Blood flow perfusion monitors the changes of cerebral blood flow perfusion in each group during reperfusion.Rats in each group were sacrificed and plasma and kidney tissues were sacrificed after reperfusion.The expression levels of BUN,Scr,IL-6 and TNF-? were measured by enzyme-linked immunosorbent assay.Morphology and structure of rat kidney tissues were observed with hematoxylin and eosin Pathological morphological changes,Sirius red staining to observe the kidney collagen deposition.Further design experiments,randomly selected 48 SPF SD male rats,the experiments were divided into 8 groups: Control + AdGFP,MCAO / R + AdGFP,Control + AdSIK2,MCAO/R + AdSIK2,Control + methyl cellulose,MCAO/R + methylcellulose,Control + Bosutinib,MCAO/R + Bosutinib,was used to detect the expression level of SIK2 by Q-PCR and Western Blot.Sirius red staining was used to observe the degree of kidney damage.LC3 A / B was used to observe the occurrence of renal autophagy in each group.Results:(1)Results of blood perfusion in pericam psi groups After ischemia for 3 hours in the experimental group,the blood perfusion of the right cerebral blood flow was significantly lower than that of the left cerebral blood flow,with an average decrease of(50.70±0.69)%.The average decrease amounts were(38.53±0.24)%,(6.04±0.03)%,(1.56±0.89)% respectively after reperfusion for 6 h,12 h and 24 h after unplugging the thread plug.Compared with 6h group,12 h group and 24 h group had difference in blood perfusion decrease(P<0.05).(2)Morphological results of HE staining groupsHematoxylin staining showed that the experimental group had kidney edema,massive red blood cell exudation,protein cast in the lumen,dilated capillary network,and increased collagen fibers.Sirius red staining showed a small amount of collagen deposition in the renal interstitium of the experimental group.(3)The expression levels of BUN,Scr,IL-6 and TNF-? in each group of ratsThe serum creatinine and urea nitrogen in the experimental group were higher than those in the sham operation group,and the difference was statistically significant(P<0.05)at 6h reperfusion compared with the sham operation group.IL-6 and TNF-? in the experimental group were both higher than those in the sham operation group,and the difference was statistically significant(P<0.05)at 6h reperfusion compared with the sham operation group.(4)Q-PCR detection of SIK2 mRNA expressionThe expression level of SIK2 mRNA was determined by Q-PCR technique during reperfusion ischemia and reperfusion.The expression of SIK2 mRNA was the lowest after 6h of reperfusion,which was statistically significant compared with the sham operation group(P <0.05).After 12 hours of reperfusion,the expression of SIK2 mRNA gradually recovered after 24 hours,and the difference was statistically significant compared with the sham operation group(P <0.05).The Western Blot technique was used to determine the expression trend of SIK2 protein at different reperfusion times.The expression of SIK2 protein was the lowest after 6 hours of reperfusion,and the difference was statistically significant compared with the sham operation group(P <0.05).After 12 hours of reperfusion,the expression of SIK2 protein gradually recovered after 24 hours,and the difference was statistically significant compared with the sham operation group(P <0.05).(5)Sirius red staining results of rats in each group Sirius red staining showed that some areas in MCAO/R+AdGFP group,MCAO/R+AdSIK2 group,MCAO/R+ methylcellulose and MCAO/R+Bosutinib group were dyed red,indicating that some collagen deposition was found in renal interstitium,while nuclei in renal tissues of Control group,Control+AdSIK2,Control+methylcellulose group and Control+Bosutinib group were dyed blue,and there was no collagen deposition in renal interstitium.(5)Results of Sirius red staining of rats in each groupSirius red staining showed that some areas in the MCAO / R + AdGFP group,MCAO / R + AdSIK2 group,MCAO / R + methylcellulose and MCAO / R + Bosutinib group were stained red,and the MCAO / R + AdSIK2 group was more stained than others.The collagen in the group was deeply stained,and the difference was statistically significant(P <0.05).The MCAO / R + Bosutinib group had lighter collagen staining than other groups,and the difference was statistically significant(P <0.05).In the Control group,Control + AdSIK2,Control + methylcellulose group,and Control + Bosutinib group,the nuclei of the kidney tissue were stained blue,and there was no collagen deposition in the renal interstitium(P> 0.05).(6)Expression trend of SIK2,CREB,LC3?/? in rats of each groupWestern blotting was used to detect the expression levels of SIK2,CREB and LC3?/? in rats of each group to explore the regulatory relationship between SIK2,CREB and LC3 ? / ?.The results showed that compared with Control group,Control+AdSIK2,Control+ methylcellulose group and Control+Bosutinib group,SIK2 level expression in MCAO/R+AdSIK2 group and MCAO/R+Bosutinib group had statistical difference(all P<0.05).There was no significant difference in CREB expression among the groups(P>0.05).Compared with Control+AdGFP group,MCAO/R+AdGFP group and Control + AdSIK2 group,the expression of MCAO/R+AdSIK2 group was significantly different(P<0.05).Compared with control+methylcellulose group,Control+bosutinib group and MCAO/R+bosutinib group,the expression of LC3? in MCAO/R+methylcellulose group was statistically different(P<0.05).Conclusion: SIK2 participates in the occurrence and development of acute kidney injury induced by cerebral ischemia-reperfusion by regulating autophagy. |
PDF Full Text Request