The Role And Mechanism Of CKMT1-mediated Mitochondrial Homeostasis And ROS Generation Via Reverse Electron Transfer In Inflammatory Bowel Disease

BackgroundInflammatory bowel disease(IBD)is a chronic,progressive inflammatory disorder affecting gastrointestinal tract,and characterized by a relapsing course.It mainly includes Crohn’s disease(CD)and ulcerative colitis(UC).In recent years,the incidence of IBD in newly industrialized countries such as China is accelerating,and the prevalence of IBD is also on the rise worldwide.IBD is increasingly becoming a heavy burden on global health.However,effective treatments are still lacking due to the extremely complex pathogenesis of IBD.We urgently need to further explore and clarify the exact mechanism of IBD pathogenesis,which may contribute to the development of new drugs for clinical treatment.Mitochondria are the central hub of energy metabolism in cell and play key roles in coordinating immunity,stress responses,and apoptosis,thus largely determining cell fate.Mitochondria has a relatively independent quality control system,which can maintain mitochondrial homeostasis via various regulatory mechanisms.Disrupted mitochondrial homeostasis will lead to mitochondrial dysfunction,which will subsequently affect a series of cell physiological processes.Previous studies have demonstrated that mitochondrial dysfunction is closely related to the pathogenesis of IBD.As a member of the creatine kinase family,CKMT1(mitochondrial creatine kinase 1)plays an important role in controlling mitochondrial energy metabolism by transferring high-energy phosphates from ATP to creatine.Thus,it acts as a key molecule in maintaining mitochondrial function.Previous studies had shown that CKMT1 regulated reactive oxygen species(ROS)production in cardiomyocytes,thus affecting the progression of heart diseases like heart failure.However,the role of intestinal CKMT1 expression in IBD remains unclear,which is worthy of further exploration.In this study,we assessed the changes of CKMT1 expression during IBD progression using colonic biopsy tissues from UC and CD patients,and colon tissues from mouse colitis models.Subsequently,the role of CKMT1 in colon inflammation and mitochondrial homeostasis was evaluated by generating intestinal epithelial-specific CKMT1 knockout(KO)mice.We then established IBD mouse model and examined the effects of CKMT1 KO on several important phenotypes such as intestinal epithelial barrier function,oxidative stress,and apoptosis.Finally,we evaluated the regulatory role of CKMT1 in mitochondrial ROS production and apoptosis,and explored its potential mechanisms.Methods1.Evaluating the expressions of CKMT1 in colonic tissues from IBD patients and experimental colitis mice models(1)Firstly,by using a public human proteome database,the expression of CKMT1(at protein and transcription levels)in human organs and tissues was investigated.The transcriptional expression of CKMT1 in human colon and small intestine tissue was also explored at the single-cell level.(2)Several healthy volunteers and active IBD patients(including UC and CD)were recruited and their colonic tissues were collected by biopsy.Then,the expression of CKMT1 in colonic tissues was detected by Western blot and immunohistochemical staining.(3)The expression of CKMT1 in colon was detected by Western blot and immunofluorescence staining in a mouse model of experimental colitis induced by dextran sulfate sodium(DSS).2.Evaluating the effects of intestinal epithelial-specific CKMT1 knockout on the colitis,mitochondrial homeostasis,oxidative stress,and apoptosis in mice(1)CRISPR/Cas-mediated genome engineering was used to create intestinal epithelial-specific CKMT1 KO mice.The growth,intestinal mucosal structure and function of the KO mice were evaluated by gross observation,histopathology and RNA-sequencing.(2)DSS and 2,4,6-trinitro-benzene-sulfonic acid(TNBS)-induced colitis were established to evaluate the effects of CKMT1 KO on colitis in mice.Intestinal epithelial barrier,inflammatory factor,and mitochondria damage of colonic tissue were assessed via several experimental techniques,such as Western blot and transmission electron microscope.(3)The effects of CKMT1 KO on mitochondrial homeostasis,oxidative stress and apoptosis of intestinal epithelial cells(IECs)in mouse colitis were evaluated by several experimental techniques like real-time fluorescent quantitative PCR,Western blot,and Tunel staining.3.The specific mechanism of CKMT1 in regulating apoptosis and mitochondrial ROS production in IECs were explored in vitro(1)Gene editing technology was applied to knockdown or overexpress CKMT1expression in cell lines.The activation of extrinsic and intrinsic apoptotic pathway were induced by TNF-αand staurosporine,respectively,to assess the effect of CMKT1 on apoptosis.The intestinal crypts of KO mice were extracted and cultured into organoids for further evaluation of the role of CKMT1 on organoid apoptosis.(2)The effect of CKMT1 on ROS production was analyzed by flow cytometry and fluorescence microscopy.A novel fluorogenic dye specifically targeted to mitochondria as well as several compounds which could disrupt mitochondrial electron transport were used to explore the source of ROS.Mitochondrial membrane potential and NDAH/NAD~+were assessed to further identity the mechanism of ROS generation.Flow cytometry and Western blot were used to evaluate the relationship between ROS and mitochondrial permeability transformation pore(m PTP),and their effects on apoptosis.Results1.CKMT1 expression was down-regulated in the colonic tissues of IBD patients and mice colitis models induced by DSS.(1)CKMT1 is highly expressed in human gastrointestinal tract(at transcription and protein levels),single-cell transcriptome data of intestinal tract tissue showed that CKMT1 was predominantly expressed in IECs.(2)The protein expressions of CKMT1 were down-regulated in colon tissues of active IBD patients(including UC and CD)and DSS-induced mice colitis.2.Intestinal epithelial CKMT1 deletion aggravated DSS-induced experimental colitis,disrupted mitochondrial homeostasis,and increased oxidative stress and apoptosis of IECs(1)Colonic CKMT1 is mainly expressed in IECs in mouse.(2)CKMT1 deficiency aggravated DSS-induced colitis in mice,worsen intestinal barrier function,and caused severe mitochondrial structural damages of IECs.(3)CKMT1 deficiency aggravated TNBS-induced colitis in mice.(4)Loss of CKMT1 in IECs resulted in imbalance of mitochondrial homeostasis,indicated by disrupted mitochondrial biogenesis,mitochondrial dynamic and mitochondrial antioxidant system.CKMT1 deficiency also aggravated oxidative stress and apoptosis of IECs.2.CKMT1 expression increased apoptosis resistance of IECs,and regulated mitochondrial reverse electron transport-derived ROS(RET-ROS)generation induced by TNF-α(1)CKMT1 expression limited activation of both intrinsic and extrinsic apoptotic pathways in IECs in vitro.(2)CKMT1 deficiency increased intestinal organoid apoptosis induced by inflammatory factors.(3)Knockdown of CKMT1 expression in IECs leaded to increased TNF-α-induced mitochondrial ROS generation,which derived from reverse electron transfer(RET).(4)RET-ROS promoted the opening of m PTP.Treatment approaches through scavenging ROS or inhibiting m PTP opening could rescue the apoptosis phenotype of IECs caused by decreased CKMT1 expression.ConclusionColonic CKMT1 expression was markedly decreased during IBD.Mice with intestinal epithelial CKMT1 deficiency showed increased susceptibility to experimental colitis.CKMT1 deficiency also aggravated mitochondrial homeostasis imbalance,oxidative stress and apoptosis of IECs.In vitro experiments further demonstrated the key role of CKMT1 expression in limiting apoptosis of IECs.Mechanisms study suggested that CKMT1 may participate in the control of IECs apoptosis via regulating TNF-α-induced RET-ROS generation and subsequent m PTP opening.In conclusion,our data demonstrated that CKMT1 is important in maintaining mitochondrial homeostasis and regulating mitochondrial ROS generation in IECs.It appears to play a key role in IBD pathopoiesis.