Dexmedetomidine Modulates Multiple Programmed Cell Death To Attanuate Acute Lung Injury Induced By Renal Ischemia-reperfusion

Acute kidney injury(AKI)induced by renal ischemia reperfusion(RI/r)is a common critical illness associated with all clinical disciplines,with an incidence of 10-15% in hospitalized patients and more than 50% in the intense care unit(ICU)patients.Although the diagnoses and therapies of AKI have been well enhanced in recent years,there is no significant improvement in the morbidity and mortality associated with it.AKI often leads to pathological injury and dysfunction of distal lung tissue,which is an important reason for its high mortality.The effects of AKI on the lungs are mainly related to two aspects: one is that acid-base imbalance,electrolyte disturbance,water-sodium retention and overload volume lead to pulmonary edema due to impaired function of glomeruli and tubules.The other is that systemic inflammatory response triggered by the nephrogenic inflammatory cytokines and danger-associated molecular patterns(DAMPs)from AKI,leads to immune cell response and secondary traumatic inflammatory response in the lung.All the above causes can lead to cell loss in lung and thus affect respiratory function.Previous studies have focused on apoptosis to demonstrate the role of upregulation of apoptosis in lung injury after AKI via some methods,such as the analysis of whole-lung gene microarrays and the detection of caspase-3 expression in the lung.Our previous studies have found Caspase-3 activation-mediated cell apoptosis of lungs in the model of RI/r-induced AKI in mice;Receptor interaction protein(RIP1)and RIP3phosphorylation-mediated necroptosis(Necroptosis),and poly ADP ribose polymerase 1(PARP-1)-dependent death(Parthanatos)have been shown in the model of renal allotransplantation in rat.Recent studies have indicated the existence of multiple patterns of cell death,which may play an important role in pulmonary dysfunction.For example,many studies have confirmed that over-activated autophagy and ferroptosis lead to lung tissue cell death via the model of lipopolysaccharide(LPS)-induced acute lung injury(ALI),thus resulting in structural destruction and dysfunction of the lung.It is of great significance to explore the multiple patterns of lung cell death and time course changes during AKI for the formulation of targeting intervention strategies.Dexmedetomidine(Dex),a highly selective α2 adrenergic receptor(α2AR)agonist,holds dose-dependent benefits such as sedation,analgesia,anxiolysis and significant reduction in postoperative delirium and agitation,and therefore is widely used in clinical anesthesia and ICU units.It has been previously shown that Dex can be protective against lung injury,but the mechanism remains unclear.Our previous studies have indicated thatα-2AR activation attenuates inflammatory response and alleviates AKI-induced ALI by ameliorating pulmonary microvascular endothelial permeability and regulating alveolar macrophages polarity switch.Some studies have shown that under certain pathophysiological conditions,Dex acts as an organ protective effect by regulating cell death.However,there is a lack of research on the pattern of lung cell death regulated by Dex under the pathological background of AKI-induced ALI.Therefore,it deserves further exploration on whether the mechanism of pulmonary protective effect of Dex is related to the regulation of multiple modes of cell death in lung.In this study,we established an animal model of ALI induced by renal ischemia-reperfusion AKI in mice based on our studies as previously described and relevant literature search and explored the time course of lung cell death pattern in RI/r48 h by detecting the levels of serum creatinine(Cr)and blood urea nitrogen(BUN)and the expression of multiple cell death pattern markers in lung tissue at different time points after reperfusion.By detecting the changes of the above-mentioned indexes after Dex pretreatment,the regulatory effect of Dex on multiple cell death modes of AKI-induced ALI was preliminarily verified and the potential mechanism of the above phenomenon was discussed.Research content1: Study on the time course changes of distal lung cell apoptosis,necroptosis,autophagy and ferroptosis caused by acute renal injury.Thirty 8-week-old male C57BL/6J mice were randomly divided into 6 groups(n=5):sham group only underwent laparotomy and closed abdomen;renal ischemia-reperfusion(RI/r)6,12,24,36 and 48 h groups,with bilateral renal ischemia for 50 min then followed by reperfusion for 6,12,24,36 and 48 h.The lung tissue was prepared for paraffin section and HE staining for pathological evaluation;after serum collection and preparation,the concentration of Cr and BUN in serum was determined by dedicated kits;the expression of Cleaved-caspase3 was detected by TUNEL staining and Western Blot to reflect the level of apoptosis in lung tissue;Immunofluorescence staining was used to detect phosphorylation mixed lineage kinase domain like protein(p-MLKL)and Western Blot to detect phosphorylated RIP1(p-RIP1)and p-MLKL to speculate the level of programmed necroptosis in lung tissue;Western Blot was used to detect the expression of p62 and LC3 to reflect autophagy in lung tissue.Western Blot and immunofluorescence staining were used to detect glutathione peroxidase 4(GPX 4)to reflect the level of ferroptosis in lung tissue.2: Regulatory effect of dexmedetomidine on multiple programmed cell death in RI/r-induced ALI.Fifteen 8-week-old male C57BL/6J mice were randomly divided into three groups(n=5):sham group was performed with laparotomy and abdomen closure only.renal ischemia-reperfusion group(RI/r),with bilateral renal ischemia for 50 min followed by reperfusion for 48 h;Dex pretreatment group(Dex+RI/r),25 μg/kg Dex 15 min prior to bilateral renal ischemia was intraperitoneally injected and the rest as before.After surgery or48 h of reperfusion,whole blood was collected to prepare serum for Cr and BUN concentrations detection;paraffin sections were prepared from lung tissues and stained with HE for pathological assessment;TUNEL was used to detect lung cell apoptosis,immunofluorescence staining to detect p-MLKL and GPX4 and Western Blot to evaluate the expression of p-RIP1,p-MLKL,Cleaved-caspase3,p62,LC3 and GPX4.Results1.Time course changes of distal lung cell apoptosis,necroptosis,autophagy and ferroptosis induced by AKI within 48 hours after renal ischemia-reperfusion1.1 Time course changes of renal and pulmonary injury after RI/rWithin 24 h after renal ischemia-reperfusion,the levels of serum Cr and BUN and lung pathological injury scores of HE staining gradually increased with time,and the degree of renal impairment was positively correlated with the degree of lung injury.1.2 Time course changes in expression of apoptosis markers after RI/rCompared with the sham group,apoptosis-positive cells in lung tissue were significantly increased in the RI/r12,24,36 and 48 h groups(p<0.05),and the expression level of Cleaved-caspase3 was significantly increased in the RI/r24,36 and 48 h groups(p<0.05).1.3 Time course changes in expression of necroptosis markers after RI/rCompared with the sham group,p-MLKL positive cells and protein expression of p-MLKL gradually increased with reperfusion time;protein expression of p-MLKL peaked at36 h of reperfusion and then decreased;compared with the sham group,the protein expression level of p-RIP1 was significantly increased in the RI/r36 and 48 h groups(p<0.05).1.4 Time course changes in expression of autophagy markers after RI/rCompared with the sham group,the expression level of LC3 in lung tissue was significantly lower in the RI/r36 and 48 h groups(p<0.05),while there was no significant difference of the expression level of P62 in lung tissue between the groups(p>0.05).1.5 Changes in expression of Ferroptosis markers after RI/rThe rate of GPX4-positive cells and protein expression levels of GPX4 were significantly reduced in the RI/r24,36 and 48 h groups compared to the sham group(p<0.05).2.Modulation of multiple programmed cell death in RI/r-induced acute lung injury of dexmedetomidine after RI/r 48 h.2.1 Effect of Dex on renal and lung injury.Compared with the sham group,the RI/r group had significant increase in pathological scores of lung injury(p<0.05)and serum Cr and BUN concentrations(p<0.05);compared with the RI/r group,the Dex+RI/r group had significant decrease in pathological scores of lung injury(p<0.05)and serum Cr and BUN concentrations(p<0.05)2.2 Effect of Dex on lung cell apoptosisCompared with the sham group,the rate of TUNEL-positive cell and the expression level of Cleaved-caspase3 in lung were significantly higher(p<0.05)in the RI/r group;compared with the RI/r group,the rate of TUNEL-positive cell and the expression level of Cleavedcaspase3 in lung tissue were significantly lower(p<0.05)in the Dex+RI/r group2.3 Effect of Dex on lung cell necroptosisCompared with the sham group,the rate of p-MLKL positive cells and the expression levels of p-MLKL and p-RIP1 in lung tissue were significantly higher(p<0.05)in the RI/r group;compared to the RI/r group,the rate of p-MLKL positive cells and the expression levels of p-MLKL and p-RIP1 in lung tissue were significantly lower(p<0.05)in the Dex+RI/r group2.4 Effect of Dex on lung cell autophagyCompared with the sham group,the LC3 expression level of lung tissue in the RI/r group was significantly lower(p<0.05),while the p62 expression level has no significant difference(p>0.05);compared with the RI/r group,the LC3 expression level of lung tissue in the Dex+RI/r group was significantly higher(p<0.05),however the p62 expression level was significantly lower(p<0.05)2.5 Effect of Dex on lung cell FerroptosisCompared with the sham group,the rate of GPX4-positive cells and the level of GPX4 protein expression in lung tissue were significantly lower(p<0.05)in the RI/r group;compared with the RI/r group,the rate of GPX4-positive cells and the level of GPX4 expression in lung tissue were significantly higher(p<0.05)in the Dex+RI/r groupConclusion1.Multiple PCD patterns,including apoptosis,necroptosis,ferroptosis and autophagy,were found in the lung of ALI induced by renal ischemia-reperfusion AKI.2.The time course changes of every PCD mode of lung cells induced by renal ischemia-reperfusion AKI are not exactly consistent.Within 48 h of renal ischemia-reperfusion,the injury of renal and lung aggravated gradually,and the levels of apoptosis and ferroptosis of lung cells increased continuously;necroptosis of lung cells peaked at 36 h and then decreased;autophagy of lung cells was significantly reduced after 36 h of renal ischemia-reperfusion.3.Dex pretreatment before renal ischemia-reperfusion can significantly attenuate renal injury and lung injury,and the amelioration of lung injury is related to its inhibition of lung cell apoptosis,necroptosis,ferroptosis and enhancement of lung cell autophagy.