Therapeutic Study Of Homologous Mitochondria On Mitochondrial Dysfunctional Liver Injury In Mice

Mitochondrial disease is caused by mitochondrial gene mutations or nuclear gene mutations,mitochondrial membrane or protein damage.It's known that it mainly affects the patient's brain,muscle,heart and so on.However,there are no specific drugs,and most of the existing treatment drugs may have many side effects.It has been reported that repair of intracellular mutations and removal of cytoplasmic protein accumulation are the fundamental methods of treating mitochondrial disease from the source of mitochondrial dysfunction.But it may have a lot of problems,such as the omplexity of operation,costly price,side effects of unknown risk,clinical application difficulties.So,this study proposes a therapeutic strategy for the replacement of dysfunctional mitochondria with biologically active mitochondria.And a simple mitochondrial injection was designed to investigate the therapeutic effect of exogenous homologous mitochondria on mitochondrial dysfunctional liver injury in mice.In this study,acetylaminophen-induced acute liver injury model and hyperlipidemic fatty liver model in mice are as mitochondrial dysfunction model of liver disease and homologous mitochondriaIn this study,homologous mitochondria were taken from Kunming mice liver.The extracted mitochondria were in good shape and the prepared mitochondrial injection was in good condition.Membrane potential ??m was measured using JC-1.Using mitochondrial selective probe MitoTracker Red CMXRos staining,mitochondria showed significant red fluorescence that demonstrated good mitochondrial integrity in the prepared mitochondrial injections.Stability of the mitochondrial injections prepared by the experiment was also determined.Results showed that the prepared mitochondrial preparations could be used for further experiments.Mitochondria stained by MitoTracker Red CMXRos and enhanced cyan fluorescent protein(ECFP)labeled mitochondrion(ECFP-Mito)were injected into the normal mice via tail vein respectivety.MitoTracker Red CMXRos lalbed mitochondria and ECFP-Mito were proved to be distributed in the whole mice body through small animal live imager.Frozen sections of the organs were to be done.Laser scanning confocal microscope(LSCM)was used to proved the distribution of MitoTracker Red CMXRos lalbed mitochondria and ECFP-Mito in the cell.Sixty healthy mice were randomly divided into 5 groups.Among them,the 4 groups were injected with mitochondria dose of 8 mg/kg as the experimental group.The fifth group was injected with 0.1 M PBS in normal control group.The levels of ATP in the tissues were measured at 1 h,8 h,24 h and 72 h respectively.Results showed that the dose of 8 mg/kg has no significant physiological toxicity and it can be used as common injection dose.Mice spontaneous activity experiments and swimming experiments further confirmed the feasibility of this dose.Mitochondria with green fluorescent protein ECFP was dubbed 0.2 mg/m L.The injection was injected into two normal mice and cells were sacrificed after 2 h.Hepatocytes were separated and the transformation of ECFP-Mito was detected by flow cytometry rate.The results show that mitochondria enter a variety of tissue cells.In the treatment of acetylaminophen-induced acute liver injury model mice,the experimental model mice were randomly divided into two groups,12 each group.In addition,12 normal mice were set to be the control.One group of APAP liver injury model group was injected with 0.8 mg/m L mitochondrial injection 10 mL/kg in the tail vein.The treatment group and the other two groups were injected with 0.01 M PBS.Levels of aspartate transaminase(AST),alanine aminotransferase(ALT)in the serum and adenosine triphosphate(ATP),glutathione(GSH)and reactive oxygen species(ROS)in liver of each mouse were measured after 12 h.Hematoxylin-eosin(HE)staining and transmission electron microscope(TEM)were taken for each group of liver.Results showed that the level of AST,ALT,ATP,ROS and GSH measured in Acetylaminophen-induced acute liver injury model were significantly different from model group and control group.HE staining showed that the model group had more severe invasive injury.Mice in the treatment group had significant improvement from the liver injury.TEM shows that mitochondria of the model group were close to the spherical shape,while the treatment group and the control group were similar to the short rod-shaped.In the treatment of mitochondria in high fat fatty liver disease model mice,the treatment group was injected mitochondrial injections twice a day for 3 times.The remaining two groups were injected PBS once every 2 days for 3 times.Levels of AST,ALT,total cholestero(TC)in serum and ATP,GSH,superoxide dismutase(SOD),malondialdehyde(MDA),triglycerides(TG)in liver of each mouse were determined on the 6th day.Liver was stained with HE,oil red O and Mayer hematoxylin staining.In the study of fatty liver model,levels of AST,ALT,TC,ATP,GSH,SOD,MDA of the therapy group were significantly difference between the model group and the control group.HE staining,oil red O and Mayer hematoxylin showed that tail vein injection of mitochondria could improve the condition of fatty liver.In summary,the mitochondrial injection prepared by this study could enter the mouse tissue after tail vein injection.Moreover,the mitochondria had no significant effect on normal mice.At the same time,it had a better effect on the acetylaminophen-induced acute liver injury model mice and high fat fatty liver model mice.Mitochondrial replacement therapy may be a new method of treating mitochondrial dysfunctional liver injury that provided a new idea for the treatment of mitochondrial diseases.