Protective Mechanism Of Dexmedetomidine On LPS-induced Acute Kidney Injury In Rats Via The Regulation Of Mitochondrial Dynamics

Acute kidney injury?AKI?caused by sepsis is a systemic infection complication with high morbidity and mortality often encountered in veterinary clinics.Due to its complicated pathogenesis,effective preventive drugs or interventions have not yet been developed.Therefore,elucidating the pathogenesis of AKI caused by sepsis and searching for effective prevention and treatment drugs have been difficult and focus of veterinary medicine and even medical research.Studies have shown that mitochondrial dynamics is an important mechanism of renal tubular apoptosis during the development of AKI.However,it is unclear whether mitochondrial dynamics imbalance mediates the occurrence of AKI caused by sepsis and its mechanism.Dexmedetomidine?DEX?has significant antioxidant,anti-apoptotic,and mitochondrial protection effects,and can also affect the regulation of mitochondrial fission/fusion.Therefore,it is speculated that DEX can play a protective role in sepsis AKI by regulating mitochondrial dynamics,and explore related mechanisms.Thirty male SD rats were selected in vivo and randomly divided into 5 groups:CON group,DEX group?30?g/kg?,and LPS group?10 mg/kg?,DEX+LPS group?30?g/kg+10 mg/kg?,Atip+DEX+LPS group?250?g/kg+30?g/kg+10 mg/kg?.After 4 h of modeling,blood was collected from the heart,urine was collected from the bladder,and kidney tissue was collected for use.Observe renal function and structure,inflammatory factors,oxidative stress,apoptosis,mitochondrial structure and function,mitochondrial dynamics,expression and localization of Ca MKII,expression and activity of Calcineurin,and expression and localization of SIRT1 and PGC-1?.In order to further reveal that mitochondrial fission mediates the occurrence of endotoxin AKI and that DEX protects endotoxin AKI by regulating mitochondrial dynamics.NRK-52E cells were selected in vitro and divided into 7 groups:CON group,LPS group?25?g/m L?,si-Drp1+LPS group,DEX+LPS group?0.001?M+25?g/m L?,si-SIRT1+DEX+LPS group,si-PGC-1?+DEX+LPS group,si-Ctrl+LPS group.Cells were knocked out of related genes with si-RNA,and after completing modeling according to the needs of each group,inflammatory factors,oxidative stress,apoptosis,mitochondrial structure and function,mitochondrial dynamics,Ca²⁺-mediated signal transduction,SIRT1/PGC-1?pathway expression.The experimental results are as follows:?1?The levels of serum BUN and Cre,urine KIM-1 and NGAL increased after LPS treatment;the above indexes were significantly reduced after DEX pretreatment;the?₂-AR inhibitor Atip significantly reversed the renal function protection of DEX,indicating that DEX can protect LPS-induced renal dysfunction by activating?₂-AR.?2?The levels of serum and cell TNF-?,IL-6,IL-1?and IL-18 were significantly increased after LPS treatment;the above indexes were significantly reduced after DEX intervention;Atip significantly reversed the anti-inflammatory effect of DEX,indicating that DEX inhibits LPS-induced inflammatory response by activating?₂-AR.After si-Drp1 inhibits the fission of Drp1,it significantly inhibits the release of inflammatory factors,suggesting that DEX is likely to inhibit the release of inflammatory factors by regulating mitochondrial dynamics.?3?Observation by light microscope,the renal cortex and medulla structure in the CON group were normal;the renal tubular dilatation,vacuole degeneration,inflammatory cell infiltration,and renal tubular necrosis in the LPS group and Atip+DEX+LPS group;mild inflammatory cells infiltration and focal necrosis in the DEX+LPS group.Transmission electron microscopy observations showed that the renal subcellular morphology of the CON group was normal;In the LPS group and the Atip+DEX+LPS group,the renal nucleus shrinks irregularly,the nuclear membrane ruptures,mitochondrial swelling,vacuolar degeneration,and the bilayer membrane structure rupture;The damage of renal subcellular organelles in DEX+LPS group was lighter than that in LPS group,and the morphology of nucleus and mitochondria returned to normal,indicating that DEX can protect the renal structure of AKI rats induced by LPS by activating?₂-AR.?4?After LPS treatment,the GSH content,CAT and SOD activities were significantly reduced,the MDA,GSSG and ROS contents were significantly increased,and the GSH/GSSG ratio was significantly reduced;after DEX pretreatment,the GSH content,CAT and SOD activities were significantly increased,and MDA,the content of GSSG and ROS decreased significantly,and the ratio of GSH/GSSG increased significantly.Atip significantly reversed the antioxidative of DEX,indicating that DEX inhibits LPS-induced oxidative stress by activating?₂-AR.After si-Drp1inhibits the fission of Drp1,it significantly inhibits the oxidative stress levels of cells,suggesting that DEX inhibit oxidative stress levels by regulating mitochondrial dynamics.?5?After LPS treatment,the apoptotic cells in renal tubules increased significantly,the expression of apoptotic proteins Bak,Bax/Bcl-2,cytoplasmic Cyt C,Cleaved caspase 9 and Cleaved caspase 3 increased,and the apoptosis genes Bak,Bax,Caspase 9 and Caspase 3 m RNA increased expression;after DEX pretreatment,the apoptosis of renal tubular cells was significantly reduced,and the expression of the above apoptotic proteins and genes were reduced;Atip significantly reversed the anti-apoptotic effect of DEX,indicating that DEX attenuates the apoptosis of renal tubular cells via mitochondrial pathway induced by LPS by activating?₂-AR.After si-Drp1 inhibits the fission of Drp1,it significantly inhibits the apoptosis of the mitochondrial pathway,suggesting that DEX may inhibit the apoptosis of renal tubular cells by regulating mitochondrial dynamics.?6?The renal mitochondrial bilayer membrane structure was complete and the crista structure was clear in the CON group;In the LPS group and the Atip+DEX+LPS group,the morphology of renal mitochondria was abnormal,obvious swelling,vacuole degeneration,bilayer membrane structure rupture and dissolution,and the crista structure was broken and blurred,mitochondrial volume became smaller;only slight adventitia dissolution was seen in the DEX+LPS group.After LPS treatment,MMP,mitochondrial respiratory chain complex I-IV activity,and ATP content were significantly reduced;MMP,mitochondrial respiratory chain complex I-IV activity,and ATP content were significantly increased after DEX pretreatment;Atip significantly reversed mitochondrial protection of DEX effect,indicating that DEX reduces LPS-induced mitochondrial structural damage and dysfunction by activating?₂-AR.After si-Drp1 inhibits Drp1 fission,it significantly inhibits LPS-induced mitochondrial fragmentation and ROS/mitochondrial ROS production,restore mitochondrial respiratory chain complex I-IV activity and MMP,enhance mitochondrial function,suggesting that DEX may inhibit mitochondrial fission,improve the structure and function of mitochondria.?7?After LPS treatment,p-Drp1 Ser616 and Fis1 protein expression were increased,Drp1 and Fis1 m RNA expression were significantly increased,Mfn1,Mfn2 and Opa1 m RNA and protein expression were decreased,Drp1,Fis1 and VDAC1 colocalization were increased;after DEX pretreatment,p-Drp1 Ser616 and Fis1 protein expression were significantly reduced,Drp1 and Fis1m RNA expression were significantly reduced,Mfn1,Mfn2,and Opal m RNA and protein expression were significantly increased,and colocalization of Drp1,Fis1,and VDAC1 were reduced;Atip significantly reversed the mitochondrial protective effect of DEX.These results indicate that DEX regulates mitochondrial dynamic balance by activating?₂-AR,inhibits mitochondrial translocation of Drp1,and protects AKI induced by LPS.After si-Drp1 inhibits Drp1 fission,it significantly inhibits mitochondrial fission and promotes mitochondrial fusion,suggesting that mitochondrial fission mediates the occurrence of AKI.?8?After LPS treatment,intracellular Ca²⁺levels were significantly increased,Ca MKII m RNA and p-Ca MKII protein expression were significantly increased,and were mainly localized in renal tubular cells;after DEX pretreatment,intracellular Ca²⁺levels were significantly reduced,Ca MKII m RNA and p-Ca MKII protein expression were significantly reduced.Atip significantly reversed the regulation of Ca MKII by DEX,indicating that DEX activates Ca MKII through?₂-AR.After si-Drp1inhibits Drp1 fission,it significantly inhibits the increase of intracellular Ca²⁺and p-Ca MKII induced by LPS,suggesting that DEX can inhibit mitochondrial fission,reduce ROS-mediated Ca²⁺overload and Ca MKII activation,and further inhibit Drp1 mitochondrial translocation to protect AKI.?9?The expressions of SIRT1 and PGC-1?m RNA and protein were significantly reduced after LPS treatment,and were mainly localized in renal tubular cells.After DEX pretreatment,the expressions of SIRT1 and PGC-1?m RNA and protein were significantly increased.Atip reversed the regulation of SIRT1 and PGC-1?by DEX,indicating that SIRT1/PGC-1?signaling pathway may participate in the protective effect of DEX on LPS-induced AKI.After si-SIRT1 and si-PGC-1?inhibit SIRT1 and PGC-1?,respectively,they significantly inhibited the protective effects of DEX on inflammatory response,oxidative stress,apoptosis,mitochondrial structure and function,and mitochondrial dynamics,suggesting that the regulation of mitochondrial dynamics by DEX at least in part via activating the SIRT1/PGC-1?pathway.In summary,inhibiting mitochondrial fission can effectively improve oxidative stress and mitochondrial dysfunction,reduce apoptosis,and thus protect the sepsis AKI.DEX can reduce the production of ROS,inhibit the Ca²⁺/Ca MKII pathway,and inhibit Drp1-mediated mitochondrial fission.DEX can also activate the SIRT1/PGC-1?pathway,thereby preventing Drp1-mediated mitochondrial fission,promoting Mfn2-mediated mitochondrial fusion,subsequently inhibiting ROS production,alleviating mitochondrial dysfunction,reducing apoptosis,and protecting sepsis AKI.These data indicate that inhibiting mitochondrial fission or maintaining mitochondrial dynamic balance can provide new therapeutic strategies for the prevention and treatment of sepsis AKI.It also provides a new molecular mechanism explanation for the protective effect of DEX on kidneys,indicating that DEX may be a promising therapeutic agent for sepsis AKI.