| BackgroundRenal ischemia/reperfusion injury(IRI)is a pathophysiological reaction of aggravated self-injury caused by the ischemic kidney regaining blood flow perfusion or oxygen supply,which may cause serious damage to renal function or even death.At present,several studies have shown that inflammatory response plays an important role in renal IRI,but the exact mechanisms is still unknown.The vagal neurotransmitter acetylcholine can regulate the inflammatory response via ?7 nicotinic acetycholine receptor(?7nAChR),alleviating the symptoms of inflammation.PHA568487 is an ?7nAChR agonist,which can activate ?7nAChR and activate the cholinergic anti-inflammatory pathway,and thus has the function of regulating inflammation in the process of organ IRI,and has broad clinical application prospects.When the body is traumatized or undergoes pathological changes that lead to cell damage,the nuclear factor-E2-related factor 2(Nrf2)will be up-regulated,which will cause oxidative stress response and ultimately promotes the cellular response to oxidative stress.In IR-induced organ damage,the activation of Nrf2 signaling pathway is dependent on the reductive coenzymes and quinone oxidoreductase to induce the inflammatory response.Nrf2 plays a protective role against various metabolic oxidative stress.Heme oxygenase(HO)is widely distributed in various tissues and organs of the body,and has the effect of protecting tissues from IRI.At present,it has been found that the activation of Nrf2/HO-1 signaling pathway is widely involved in the anti-oxidative stress damage of the heart,brain,kidney and nervous system and other tissues and organs,which can protect cells from oxidative stress damage,exert anti-oxidation and anti-apoptotic effect.During renal IRI,it is unclear whether PHA568487 can regulate the Nrf2/HO-1 pathway.Circular RNA(circRNA)is a non-coding RNA composed of end-to-end without 5'cap-like structure and 3'adenylate tail.It has the special fuction of regulating microRNA(miRNA).In recent years,more and more literatures have demonstrated that circRNA has the function of regulating various inflammation-related human diseases.CircRNA can not only be used as a biomarker to predict kidney-related diseases,but also can regulate the progression of disease by adsorbing those with a targeted relationship.In addition to the traditional inflammatory pathways,it is not clear whether PHA568487 can participate in the renal IRI inflammation process by regulating circRNA.AimThe purpose of this study was to investigate whether PHA568487 can reduce the inflammatory response in renal IRI rats,and to analyze the internal regulatory mechanisms that maybe involved.An animal model of rat kidney IRI was established and then the effects of PHA568487 pretreatment on the serum urea nitrogen,serum creatinine,serum inflammation-related cytokines,renal pathological changes of rat kidney tissue and apoptosis of kidney tissue were detected to analyze whether PHA568487 pretreatment has a relieving effect on rat kidney IRI.On this basis of research,we further explore the inner mechanisms through the cell biology experiment and provide an important evidence for clinical drug therapy and targeted therapy.Method1.First,we constructed Wista rat animal model of renal IRI by reperfusion-ischemia in rat kidney tissue,and established PHA568487 pretreated Wista rat kidney by injecting 2.4 mg/kg PHA568487 for 2 h before reperfusion.The we observed the general state of rats in each group and collect blood samples and kidney tissue samples of rats.The levels of serum urea nitrogen and blood creatinine were measured using a blood biochemical detector,and enzyme-linked immunosorbent assay(ELISA)was used to detect C-reactive protein(CRP),Expression of tumor necrosis factor ?(TNF-?),interleukin-1?p(IL-1?)and IL-6.Subsequently,pathological sections of kidney tissues were constructed and stained with Periodic Acid-Schiff stain(PAS)to observe the pathological changes of kidney tissues of rats.TUNEL method was used to detect the apoptosis of rat kidney tissue.qRT-PCR and Western Blot were used to detect the expression of Bax,Bcl-2,Caspase-3 mRNA and protein in rat kidney tissue of each group.2.First,we constructed a model of renal IRI by placing human renal tubular epithelial cells HK-2 under hypoxic-reoxygenation environment.PHA568487 or Nrf-2 specific inhibitors ML385 was utilized to pre-treat with HK-2 and then the IRI model was established.Subsequently,we used the CCK-8 method to detect the proliferative activity of each group,and flow cytometry was used to detect the apoptosis rate of each group.Finally,total RNAs and total proteins of cells were extracted,and then the mRNA and protein expressions of Nrf-2,HO-1,Caspase-3,Bax,and Bcl-2 were detected by qRT-PCR and Western Blot,respectively.3.First,we constructed a model of renal IRI by placing human renal tubular epithelial cells HK-2 under hypoxic,glucose-depleted and reoxygenated environments PHA568487 group was constructed by the pre-treatment of HK-2 cells and IRI model was established.qRT-PCR was used to detect the expression of circYAP1 in IRI-induced HK-2 cells after PHA568487 treatment.Subsequently,HK-2 cells were transected the overexpressing circYAP1 plasmid and constructed an IRI cell model,the effect of circYAP1 on cell viability and apoptosis was detected using CCK-8 method and flow cytometry.Then,ELISA and reactive oxygen species(ROS)detection tests were conducted to detect the effects of circYAP1 overexpression on IL-1?,IL-6 secretion and ROS formation.Next,using online bioinformatics databases to predict miRNAs that have a targeted binding with circYAP1.qRT-PCR was used to detect the expression of miR-21-5p in IRI-induced HK-2 cells after circYAP1 knockdown,and luciferase reporter gene system was further used to analyze the binding of circYAP1 to miR-21-5p.Furthermore,we simultaneously upregulated circYAP1 and miR-21-5p and then construct an IRI cell model to examine the effects of miR-21-5p on cell viability,apoptosis,inflammatory factor secretion and ROS formation.Finally,the expression of PI3K/AKT/mTOR signaling pathway-related proteins in IRI HK-2 cells were detected using Western Blot experiments.Result1.In the Wista kidney IRI rat animal model,it was observed that the appetite,eating and drinking of rats was reduced and the depressed rats were less active.The blood biochemical test results show that the serum urea nitrogen and creatinine levels in rats are significantly increased.In addition,PAS staining showed the increased glomerular volume in rat kidney tissue.A large number of inflammatory cell infiltration in the interstitial area,and glomerular degeneration of glomerular cells were observed.In addition,the levels of CRP,TNF-?,IL-1?,and IL-6 in the serum of kidney IRI rats were significantly higher than those of untreated rats.The apoptosis rate of kidney tissue in IRI group rats was significantly higher.Bcl-2 mRNA and protein expression levels was significantl decreased while Bax and Caspase-3 expression levels significantly increased.Unlike the IRI group of rats,the Wista kidney IRI group pretreated with PHA568487 had better drinking water,food intake,mental state and activity.The serum urea nitrogen and creatinine levels were reduced,and the renal tissue pathological status was obvious improved.This demonstrats that PHA568487 can significantly improve renal tissue lesions in rats with renal ischemia-reperfusion injury.In addition,after pretreatment with PHA568487,the levels of CRP,TNF-?,IL-1?,IL-6 and the apoptosis rate of tissue cells in renal IRI rats were significantly reduced,and the expression of anti-apoptosis genes was increased and the pro-apoptosis gene expression is inhibited.2.In the HK-2 cell model simulating kidney IRI,the cell proliferation activity was lower than that of the control group,but the apoptosis rate was higher,accompanied by a decrease in the expression levels of Nrf2,HO-1,Bcl-2 and Caspase-3 and Bax expression levels.After PHA568487 pretreatment,the proliferative activity and apoptosis rate of HK-2 cells in renal IRI is increased,and PHA568487 can also increase the expression level of Nrf2,HO-1,Bcl-2 in HK-2 cells of renal IRI and inhibit Caspase-3 and Bax mRNA and protein expression.In addition,unlike the PHA568487 alone pretreatment group,the cell proliferation activity and the apoptosis rate of the group incubated with PHA568487 and Nrf2 inhibitor were significantly reduced,and the expression levels of Nrf2,HO-1,Bcl-2 mRNA and protein were significantly increased.The expression level of Caspase-3 and Bax mRNA and protein was significantly increased.3.Compared with HK-2 cells of IRI,PHA568487 pretreatment can increase the expression level of circYAP 1 in HK-2 cells.By transfecting the overexpressing c irc YAP 1 plasmid into HK-2 cells and constructing an IRI cell model,the results of CCK-8 and flow cytometry showed that after overexpression of circYAP1,the cell viability was enhanced and the apoptosis ability was weakened.Moreover,IL-1? and IL-6 secretion and ROS generation was enhanced.These data indicates that up-regulating circYAP1 expression can alleviate the inflammatory damage of HK-2 cells.Bioinformatics database and qRT-PCR experiments showed that there are multiple miRNAs with circYAP1 targeting complementary sequences,and these miRNA expression levels increased in the circYAP1-silenced IRI cells.Subsequently,we verified that circYAP1 acts as a sponge for miR-21-5p using luciferase reporter gene assay.In addition,further cell biology experiments also showed that miR-21-5p may be involved in the inflammatory injury process which was regulated by circYAP1 in HK-2 cells.In other word,up-regulating miR-21-5p reversed the mitigating effect of circYAP1 overexpression on inflammation damage.Finally,Western Blot showed that overexpression circYAP1 promoted the expression of phosphorylated PI3K,AKT,and mTOR proteins by inhibiting miR-21-5p expression levels,thereby activating PI3K/AKT/mTOR signaling pathway.ConclusionThis study focused on the effects of PHA568487 pretreatment on renal IRI cell apoptosis,inflammatory response,and possible mechanisms of action.A series of experiments were carried out,and the following conclusions were obtained:1.PHA568487 pretreatment can effectively protect renal function damage caused by ischemia-reperfusion and inhibit renal tissue cell apoptosis;2.The protective effect of PHA568487 pretreatment on renal IRI is related to its ability to inhibit the expression of inflammatory factors,inhibit the expression of pro-apoptotic genes,and promote the expression of apoptosis-inhibiting genes;3.The Nrf-2/HO-1 pathway promotes the expression of the apoptosis inhibitory gene Nrf2 by inhibiting the expression of the pro-apoptotic genes Caspase-3 and Bax,and then exerts its role in inhibiting the apoptosis of HK-2 cells of IRI.The effect of PHA568487 on HK-2 cells was partly realized by Nrf-2/HO.4.In HK-2 cells of IRI,PHA568487 pretreatment can increase the expression level of CircYAP1.By targeting miR-21-5p,CircYAP1 reduces the IRI response of HK-2 cells,promotes apoptosis of injured cells,and activates the PI3K/AKT/mTOR signaling pathway. |
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