Protective Effect Of Coenzyme Q10 On Acute Lung Injury Mice By Regulating MKP-1/Nrf2/Drp1 Signaling Pathway

Acute lung injury(ALI)is a common and life-threatening respiratory disease in veterinary clinic.In view of its high morbidity and mortality,unclear pathogenesis and imprecise treatment,in-depth research on the molecular pathogenesis of ALI and the development and exploration of effective therapeutic drugs are urgent problems to be solved in the prevention and treatment of animal respiratory diseases.Recent studies have found that mitochondrial damage is an important pathogenesis of ALI.However,the underlying molecular mechanisms by which the dynamic imbalance of mitochondrial fission and fusion mediates ALI in mice remains inconclusive.Coenzyme Q10(CoQ10)is an endogenously synthesized and highly safe fat-soluble compound without obvious toxic and side effects.It has powerful antioxidant,anti-inflammatory,anti-apoptotic and lung protective properties,and can restore mitochondrial metabolic enzyme activity and improve mitochondrial damage.Therefore,it is speculated that CoQ10 can protect mouse ALI by regulating the dynamic balance of mitochondrial fission and fusion.In order to explore the effect and intervention mechanism of CoQ10 on LPS-induced ALI in mice,126 male C57BL/6 mice were selected and randomly divided into CON group(100 μL corn oil + CON),CHQ group(50 mg/kg CoQ10 + CON),LPS group(100 μL corn oil + LPS),LDQ group(2 mg/kg CoQ10 + LPS),LMQ group(10 mg/kg CoQ10 + LPS)and LHQ group(50 mg/kg CoQ10+ LPS).After continuous intraperitoneal injection of CoQ10 or corn oil for 14 d,the mouse ALI model was established by intranasal instillation of LPS(50 μg/50 μL).By observing the clinical symptoms,lung coefficient,wet to dry weight ratio(W/D),BALF inflammatory cell and protein concentration,lung anatomy and microstructure,pulmonary surfactant and AQP5 content,lung epithelial barrier structure factor expression,inflammatory factor levels,MPO activity,NLRP3 inflammasome expression,oxidative stress,apoptosis,mitochondrial structure and function,mitochondrial fission and fusion,MKP-1/Nrf2 signaling pathway-related gene and protein expression,to clarify the effect and mechanism of different doses of CoQ10 on LPS-induced ALI in mice,and screen out the best protective dose of CoQ10.Then,another 126 male C57BL/6 mice were selected and randomly divided into CON group(100 μL corn oil + CON),LPS group(100 μL corn oil + LPS),LQ group(50 mg/kg CoQ10 + LPS),LS group(100 μL corn oil + 160 mg/kg Schaftoside + LPS),LMK group(50 mg/kg CoQ10 + 4 mg/kg NSC 95397 + LPS)and LN group(50 mg/kg CoQ10 + 2 mg/kg Brusatol + LPS).The mouse ALI model was established by continuous intraperitoneal injection of CoQ10 or corn oil for 14 d,and intranasal instillation of LPS(50 μg/50μL).By observing MKP-1/Nrf2 signaling pathway-related gene and protein expression,mitochondrial fission and fusion,mitochondrial structure and function,apoptosis,oxidative stress,NLRP3 inflammatory response,lung epithelial barrier structural factor expression,pulmonary surfactant and AQP5 expression,lung anatomy and microstructure,BALF inflammatory cell and protein concentration,lung coefficient,W/D and clinical symptoms of mice,to clarify the pathological mechanism of mitochondrial dynamic imbalance mediating ALI in mice and the molecular mechanism of CoQ10 intervention.(1)The observation test results of the protective effect of different doses of CoQ10 on ALI mice showed that:1)Different doses of CoQ10 can gradually improve the symptoms of depression,coarse hair and tachypnea in ALI mice,and reduce pulmonary coefficient,W/D value,BALF inflammatory cell and protein concentration,indicating that CoQ10 can reduce pulmonary edema,alleviate the permeability of pulmonary epithelium and capillary endothelial in a dose-dependent manner,and protect ALI mice.2)Lung swelling,pallor,alveolar wall thickening,lung tissue bleeding,inflammatory cell infiltration,and alveolar transparent membrane formation in LPS group.After treatment with different doses of CoQ10,the above phenomena were gradually reversed,indicating that CoQ10 improved the lung histopathological injury of ALI mice in a dose-dependent manner.3)Different doses of CoQ10 can gradually reverse the LPS-induced reduction of pulmonary surfactant,AQP5 and lung epithelial tight junction structure factor m RNA expression,qualitative expression of tight junction factor,β-catenin and E-cadherin co-localization and protein expression.These results showed that CoQ10 improved the structural and functional damage of pulmonary epithelial barrier in ALI mice in a dose-dependent manner.4)Different doses of CoQ10 can gradually reduce the concentrations of TNF-α,IL-6,IL-1βand IL-18 in the lung tissue and serum of ALI mice,inhibit the m RNA and protein expressions of inflammatory factors,and attenuate MPO activity,NLRP3 inflammasome m RNA and protein expression and co-localization of NLRP3 with mitochondria,indicating that CoQ10 attenuated NLRP3 inflammation in ALI mice in a dose-dependent manner.5)Different doses of CoQ10 can gradually reduce the content of MDA and ROS in the lung tissue of ALI mice,enhance the activities of SOD and CAT,and increase the level of GSH,indicating that CoQ10 can inhibit oxidative stress in ALI mice.6)Different doses of CoQ10 can gradually reduce TUNEL-positive cells in ALI mice,inhibit the expression of Bax,Bax/Bcl-2,Cyt-c,Caspase-9 and Caspase-3 gene and protein,promote the expression of Bcl-2 gene and protein,and prevent the co-localization of Bax with mitochondria.The results showed that CoQ10 ameliorated mitochondrial apoptosis in ALI mice in a dose-dependent manner.7)In the LPS group,the lamellar bodies of mouse lung cells vacuolated,the parallel layers disappeared,the mitochondria swelled and ruptured,the mitochondrial crists dissolved and disappeared,there were a large number of mitochondrial ghosts,nuclear pyrosis,and nuclear membrane rupture.The mitochondrial membrane potential,complex I-IV activity and ATP content decreased,while mt DNA and mt ROS levels increased.These phenomena were gradually reversed by different doses of CoQ10,suggesting that CoQ10 improved mitochondrial structure and function damage in a dose-dependent manner.8)CoQ10 can inhibit the expression of P-Drp1(ser 616)protein,Fis1 m RNA and protein,and Drp1 and Fis1 co-localization with mitochondria,and promote the expression of OPA1,Mfn1 and Mfn2 gene and protein.These results suggested that the protective effect of CoQ10 on ALI mice might be attributed to its blocking of mitochondrial division and driving mitochondrial fusion.9)CoQ10 can promote the m RNA and protein expression of MKP-1,Nrf2,HO-1 and NQO1 in the lung tissue of ALI mice,indicating that the protection of ALI by CoQ10 may be related to the activation of MKP-1 and Nrf2 signaling pathways.(2)The experimental results of the mechanism of CoQ10 protecting ALI mice showed that:1)CoQ10 and Drp1 inhibitors reversed LPS-induced MKP-1,Nrf2,HO-1 and NQO1 m RNA and protein expressions in mouse lung tissue,and MKP-1 inhibitors blocked the effect of CoQ10 on MKP-1 and Nrf2.Nrf2 inhibitor attenuated the m RNA and protein expressions of Nrf2,HO-1 and NQO1,but had no effect on the m RNA and protein expressions of MKP-1,indicating that CoQ10 plays a protective role in ALI mice by up-regulating MKP-1 and then activating the Nrf2 signaling pathway.2)CoQ10 and Drp1 inhibitors reversed the LPS-induced increases in P-Drp1(ser 616)protein expression,Fis1 gene and protein expression,and colocalization of Drp1 and Fis1 with mitochondria in mouse lung tissues.In addition,they also enhanced the expression of OPA1,Mfn1 and Mfn2 genes and proteins.MKP-1 and Nrf2 inhibitors both blocked the regulatory effect of CoQ10 on mitochondrial fission and fusion,indicating that CoQ10 inhibited mitochondrial fission and promoted mitochondrial fusion by activating the MKP-1/Nrf2 signaling pathway.3)CoQ10 and Drp1 inhibitors reversed LPS-induced vacuolization of lamellar bodies,mitochondrial swelling,cristae dissolution and disappearance,pyknosis,nuclear membrane rupture,decreased mitochondrial membrane potential,complex I-IV activity and ATP content,and increased mt DNA and mt ROS levels.Both MKP-1 and Nrf2 inhibitors blocked the protective effect of CoQ10 on mitochondria,indicating that CoQ10 can improve mitochondrial structure and function damage in ALI mice by activating the MKP-1/Nrf2 signaling pathway and inhibiting mitochondrial fission.4)CoQ10 and Drp1 inhibitors reversed the LPS-induced increase in TUNEL-positive cells and Bax,Bax/Bcl-2,Cyt-c,Caspase-9 and Caspase-3 m RNA and protein expressions,and Bcl-2 m RNA and protein expressions.expression decreased.Both MKP-1 and Nrf2 inhibitors blocked the antiapoptotic effect of CoQ10,indicating that CoQ10 attenuated apoptosis by activating the MKP-1/Nrf2 signaling pathway and inhibiting mitochondrial fission.5)CoQ10 and Drp1 inhibitors decreased MDA and ROS contents,enhanced SOD and CAT activities,and increased GSH levels.Both MKP-1 and Nrf2 inhibitors blocked the antioxidant effect of CoQ10,indicating that CoQ10 attenuated pulmonary oxidative stress by activating the MKP-1/Nrf2 signaling pathway and inhibiting mitochondrial fission.6)CoQ10 and Drp1 inhibitors reduced the levels of TNF-α,IL-6,IL-1β and IL-18 in lung tissue and serum,inhibited inflammatory factors m RNA and protein expressions,attenuated MPO activity,NLRP3 inflammasome m RNA and protein expression,and NLRP3 co-localization with mitochondria.Both MKP-1 and Nrf2 inhibitors blocked the anti-inflammatory effect of CoQ10,indicating that CoQ10 attenuated lung inflammation by activating the MKP-1/Nrf2 signaling pathway and inhibiting mitochondrial fission.7)CoQ10 and Drp1 inhibitors both increased pulmonary surfactant and AQP5 and lung epithelial tight junction structural factor m RNA expression,tight junction structural factor qualitative expression,β-catenin and E-cadherin co-localization and protein expression.Both MKP-1 and Nrf2 inhibitors reversed the above phenomenon,indicating that CoQ10 improved the structure and function of the lung epithelial barrier in ALI mice by activating the MKP-1/Nrf2 signaling pathway and inhibiting mitochondrial fission.8)CoQ10 and Drp1 inhibitors improved LPS-induced lung anatomical and histological damage.Both MKP-1 and Nrf2 inhibitors reversed the lung tissue protective effect of CoQ10,indicating that CoQ10 ameliorated lung histopathological damage in ALI mice by activating the MKP-1/Nrf2 signaling pathway and inhibiting mitochondrial fission.9)CoQ10 and Drp1 inhibitors ameliorated LPS-induced depression,coarse hair and tachypnea in mice,and decreased lung coefficient,W/D value,and BALF inflammatory cell and protein concentrations.MKP-1 and Nrf2 inhibitors reversed the above phenomenon,indicating that CoQ10 could protect ALI in mice by activating the MKP-1/Nrf2 signaling pathway,inhibiting mitochondrial fission,attenuating pulmonary edema,relieving lung epithelial and capillary endothelial permeability.In conclusion,LPS induced ALI in mice mainly by disrupting the dynamic balance of mitochondrial division and fusion,impaired mitochondrial structure and function,induced mitochondrial endogenous apoptosis,oxidative stress and NLRP3 inflammation,and destroyed the pulmonary epithelial barrier structure.CoQ10 protected against LPS-induced mouse ALI in a dosedependent manner,by activating the MKP-1/Nrf2 signaling pathway,increasing the expression of HO-1 and NQO1,inhibiting the phosphorylation of Drp1 616,blocking mitochondrial fission,driving mitochondrial fusion,alleviating mitochondrial damage,reducing apoptosis,oxidative stress and NLRP3 inflammation,promoting the remodeling of lung epithelial barrier structure and the production of pulmonary surfactant and AQP5,and attenuating pulmonary edema and lung tissue damage.The optimal protective dose of CoQ10 was 50 mg/kg.