| Background: Renal ischemia-reperfusion injury(IRI)is widespread in clinical practice,from embolic or thrombotic events,circulatory shock and hypotension to surgery including cardiac bypass surgery,mandatory kidney injury associated with organ harvesting,and other kidney damage.IRI may lead to renal disfunction,increasing the incidence and mortality of end-stage renal disease in patients.The short-and long-term prospects of patients depend on the reversibility of the injury and subsequent recovery.Kidneys are very sensitive to IRI,and the mismatch in oxygen supply and demand causes a decrease in oxidative metabolism in the kidneys,which leads to damage and death of tubular epithelial cells.Renal tubular epithelial cell apoptosis is a main feature of renal I/R injury,accompanied by a rapid loss of cell membrane potential,then leading to AKI.Studies have shown that mitochondria are focal points for apoptosis signals involved in the pathogenesis of kidney disease.In addition,mitochondrial damage has been reported as the main mechanism leading to renal I/R injury,and the BCL-2 family is considered to be a major regulator of the mitochondrial pathway in renal I/R injury.Caspase-3 has been used as an important regulatory site for the regulation of renal tubular epithelial cell apoptosis,and is also an important target for the protective treatment of IRI.Many researchers believed that reducing the activation of Caspase-3 is one of the effective means to protect kidney function after renal I/R.Mitochondrial apoptosis pathway is one of the important pathways of renal tubular epithelial cell apoptosis.The ratio of Bax/Bcl-2 to an equilibrium state is an important indicator for detecting the apoptosis of renal tubular epithelial cells.Activation of PI3K/AKT signaling pathway is an important protective mechanism in renal I/R injury,mainly through regulation of downstream target proteins(such as heme oxygenase(HO)-1,endothelial nitric oxide synthase(e NOS),transcription Factor NF-E2 related factors)regulate pathological damage processes such as apoptosis,inflammatory response and oxidative stress.Studies have shown that receptor-mediated PI3K/AKT activation could effectively alleviate I/R-induced renal tubular epithelial cell apoptosis and oxidative stress,and its renal protective function can be significantly reversed by PI3K/AKT inhibitors.This suggested that selective intervention of the PI3K/AKT pathway may be an effective target for the prevention and treatment of renal IRI.Apigenin(API)is a natural flavonoid,which was commonly used in nature to resist lipid peroxidation.It is found in fruits and plants(various fruits,vegetables,beans and tea,Scrophulariaceae,water vine,medlar Polygonum cuspidatum,Liliaceae,and higher content in chamomile).In addition,API possessed the activities of anti-oxidation,anti-mutation and anti-tumor including prostate cancer,colon cancer,ovarian cancer,and breast cancer.API plays its effects of anti-inflammatory,anti-apoptosis,regulation of immunity,prevention and treatment of vascular sclerosis,hypoglycemia though inhibiting COX-2 activity and reducing the production of pro-inflammatory cytokines.Compared with other flavonoids(quercetin,myricetin),API has low toxicity and no mutagenicity.The literature reports that API has protective effects on myocardial,cerebral and hepatic ischemia-reperfusion injury,drug-induced liver and kidney damage,but has not been reported on renal IRI.This experiment confirmed that API could regulate the apoptosis of renal tubular epithelial cells and has a protective effect on rat renal IRI.Objective: An animal model of acute kidney injury induced by renal artery IRI in rats was tested for the protective effect of API on renal IRI in rats.Preliminaryly,API can reduce the apoptosis of renal tubular epithelial cells to protect kidney injury;establish the hypoxia/reoxygenation model by culturing HK-2 cells to further confirm the protective effect of API on renal tubular epithelial cells,and further explore the possiblebiological mechanism of API on Renal ischemic perfusion injury.Methods: Animal experiments: 18 male Sprague-Dawley rats(weight 180 ± 10 g)were randomly divided into 3 groups: sham operation group(sham group),ischemia-reperfusion group(IR group),API pretreatment + ischemia-reperfusion group(IR+API group).The sham group was injected 0.9% saline intraperitoneally 1 hour before the operation;the IR group was not administered;the API(20 mg/kg)was intraperitoneally injected 1 hour before the IR+API group.The renal ischemia-reperfusion model was established by the method of renal pedicle clamping.The changes of renal function(creatinine and urea nitrogen)were detected by Johnson& Johnson Biochemical Autoanalyzer.The histopathology of the IR group and API treatment was observed by PAS staining.Morphological changes and injury scores was observed and recorded;the apoptosis of renal tubular epithelial cells in each group was observed by Tunnel method.Cell experiments: Renal tubular epithelial cells(HK-2)in logarithmic growth phase were selected and seeded in 6-well plates at a density of 2*105 cells per well.After 20 hours of inoculation,the cell culture medium was replaced with DMEM medium containing 1% fetal bovine serum for 24 hours to achieve the purpose of relative synchronization of cells,and hypoxia/reoxygenation model was established according to the method given in the literature.Grouping: control group: cells were cultured in normal environment;hypoxia-reoxygenation(H/R)group: treated by model establishment method;API(H/R+API)group: API 20um/l was added 2 hours before model establishment to the hypoxia/reoxygenation model.The activity of HK-2 cells in each group was detected by CCK-8 kit.The effect of API on the activity of HK-2 cells was observed.The apoptosis of each group was detected by flow cytometry.Results: The rat model of renal ischemia-reperfusion was established.The changes of renal function(Scr and BUN)in rats were detected by Johnson & Johnson BiochemicalAuto-analyzer 24 hours after operation.In the IR group,serum creatinine was79.8±8.6umol/l,urea nitrogen was 23.8±3.7mmol/l,IR+API group had serum creatinine of 39.5±6.4umol/l,and urea nitrogen was 12.6±1.2mmol/l.Creatinine and urea nitrogen levels in the IR+API group were significantly decreased.That indicated that API could protect kidney injury after ischemia-reperfusion in rats.The pathological damage was observed by PAS staining.The tissue injury was observed in IR group.The most common pathological changes were cortical and medullary junctions.The main pathological changes were interstitial hemorrhage,edema,inflammatory cell infiltration,tubular swelling,abnormal renal tubular epithelial cells,brush-like disorder disappeared,and detached tubule cells were observed in the lumen.A variety of tube types such as protein tube type,particle tube type,and red blood cell tube type can be seen.The pathological damage in the IR+API group was alleviated than that in the IR group: less interstitial hemorrhage,less edema,less infiltration of inflammatory cells,less various tube types,and alleviate abnormal morphology.It indicated that API can alleviate the damage after renal ischemia-reperfusion in rats and protect renal function.The number of apoptotic cells in the renal tubular epithelial cells of the IR group was compared with the number of positive cells in the IR+API group,and the number of apoptotic cells in the IR+API group was significantly reduced.It indicated that API treatment can inhibit the small cell epithelial apoptosis after ischemia-reperfusion injury and reduce renal tubular injury.The CCK-8 experiment showed that the cell survival rate of H/R group was 0.61±0.06,and that of H/R+API group was 0.85±0.05.The results showed that API could protect HK-2 cell activity;the apoptosis rate of HK-2 cell between control,H/R group and H/R+API group was detected by the flow cytometry Annexin IV/PI.The apoptosis rate of H/R group was 40.19%,and the apoptosis rate of H/R+API group decreased to20.98%,indicating that API can effectively inhibit the apoptosis of HK-2 cells.The activity of caspase-3 in H/R group was significantly higher than that in control group,and the expression of caspase-3 protein was significantly increased.After API intervention,Caspase-3 activity was higher than that H/R group.The expression levels of bax,Bcl-2,PI3 K,p-AKt and AKT protein were detected.After H/R injury,the expression of proapoptotic protein Bax increased,and the expression of apoptosis-inhibiting protein Bcl-2 was decreased.In addition,the expression of phosphorylated PI3 K protein was significantly decreased and p-AKT was also decreased.After the intervention of API,the Bax protein in the API group was decreased compared with the H/R group,and the expression of Bcl-2 and p-PI3 K and p-AKt protein were increased.Conclusion: This experiment demonstrates that API has a protective effect on renal ischemia-reperfusion injury by reducing apoptosis of renal tubular epithelial cells and anti-apoptotic effect of API is mainly achieved by regulating the expression and activation of Bcl-2 family proteins through mitochondrial pathway.Activation of PI3K/AKT signaling pathway plays a role in the protective effect of API on hypoxia/reoxygenation injury in HK-2 cells.However,whether API has effects on other apoptosis-related genes and related mechanisms,more comprehensive and in-depth researchs would still be needed. |
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