Exogenous Mitochondria Corrects Carbon Tetrachloride-induced Liver Fibrosis And Its Therapeutic Mechanism

Liver fibrosis can be induced by a variety of pathogenic factors(viruses,chemical poisons,obesity,etc.).It is a type of tissue damage that occurs after the healing of tissue wounds,manifested by abnormal hyperplasia of connective tissue in the liver.With the continuous development of the condition,it will further cause liver cirrhosis and even cancer,which seriously affects the physical health.During liver fibrosis,extensive mitochondrial damage occurs in liver cells,manifested as blocked energy production,massive release of oxygen radicals,altered autophagy function,and the damage of mitochondrial DNA,which are important factors causing liver dysfunction.Therefore,from the perspective of correcting the damaged state of liver mitochondria,this study attempts to use normal mitochondria to replace the defective mitochondria to restore its activity and improve liver function.The carbon tetrachloride,a chemical poison,was used to induce the fibrosis of liver(carbon tetrachloride can attack cell membranes,lead to oxidative stress dependent liver cell damage,and activate extracellular matrix hyperplasia,mediated liver fibrosis).The therapeutic drug is extracted from the liver of the identical species of healthy mice to obtain mitochondria with high-purity and good activity.In this research,the mitochondria extracted from this allogeneic are called exogenous mitochondria.In the in vitro experiments the way in which exogenous mitochondria enter hepatocytes was primarily explored.The fluorescent dye labeled exogenous mitochondria was used for mitochondria tracking.It was found that when the liver cell was pretreated with macropinocytosis inhibitor amiloride,the amount of hepatic mitochondria taken up by the liver cell was significantly reduced.Therefore the way that the exogenous mitochondria entered the liver cell may be the cellular endocytosis of macropinocytosis.Next,in order to explore the therapeutic effect of exogenous mitochondria on the damaged hepatocytes,the hepatocyte cells were incubated for 8 hours using carbon tetrachloride to induce a cell damage model.After supplementing the damaged liver cells with normal exogenous mitochondria,the recovery of cell and mitochondrial functions was detected.The results show that exogenous mitochondrial treatment for 2-4 h and a concentration between 200-400 μg/mL have a significant therapeutic effect on damaged hepatocytes.The therapeutic effect is concentration-dependent,higher concentration of mitochondrial,leads to increasing effect on the cell recovery;at the same time,it was also found that exogenous mitochondria can reduce the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the culture medium,and increase the intracellular adenosine triphosphate(ATP)level,proving that exogenous mitochondria can reduce Hepatocyte injury,restore the physiological function of mitochondria in liver cells.In the in vivo experiments,we firstly illustrated the distribution of exogenous mitochondria in the body post tail vein injection.The model of liver fibrosis was induced by long-term intraperitoneal injection of carbon tetrachloride into healthy mice.Exogenous mitochondria labeled with fluorescent dyes were injected into fibrosis model mice and normal mice.After 2 hours,the exogenous mitochondria were distributed in heart,liver,lung,kidney and brain.The results showed that exogenous mitochondria were most distributed in liver tissues.Notably,compared with non-damaged normal livers,fibrotic livers were more likely to acquire more exogenous mitochondria.In addition,the experiment also investigated the effect of short-term single dose of exogenous mitochondria on the activity of mitochondria in the damaged liver.Two hours after injection of exogenous mitochondria into liver fibrotic mice,the mitochondrial membrane potential and succinate dehydrogenase activity in the liver were detected.It was found that exogenous mitochondria significantly increased succinate dehydrogenase activity and slightly increased mitochondrial membrane potential compared with the fibrosis model group.The above results suggest that mitochondrial therapy is feasible in reducing liver fibrosis and restoring mitochondrial function.In order to further explore the therapeutic effect of exogenous mitochondria in liver fibrosis diseases,the experiment used exogenous mitochondria to carry out intervention therapy on liver fibrosis mice for one week.The results show that after injection with functional mitochondria,the liver coefficient decreases.The results showed that exogenous mitochondria alleviated liver congestion and edema caused by excessive fiber deposition.In addition,after the mitochondrial treatment,the normal liver morphology was restored.The liver surface gradually changed from rough to smooth.And the liver inside substantial cell necrosis reduced,inflammatory infiltration relieved,and the degree of fibrosis decreased.Through the blood indexes detection in mice,it was found that the level of ALT decreased in the serum after mitochondrial treatment,indicating that the degree of liver damage in mice was reduced.The mouse liver tissue homogenate was also detected.The result shows that the level of superoxide dismutase(SOD)increased and the level of reactive oxygen species(ROS)and hydroxyproline(HYP)decreased in the liver after mitochondrial treatment,indicating that the exogenous mitochondria reduced the degree of liver fibrosis and improved the ability of scavenging oxygen free radicals.The characteristic staining of collagen fiber deposits with Sirius Red further proved that exogenous mitochondria played a benign role in reducing liver fibrosis.Observing the ultrastructure in the liver through electron microscopy showed that the amount of liver mitochondria increased significantly after exogenous mitochondria treatment.In comparison,the number of swollen mitochondria decreased,indicating that exogenous mitochondria improved the state and number of mitochondria in cells.In order to deeply explore the mechanism of exogenous mitochondria in liver diseases induced by carbon tetrachloride.We compared the transcriptome differences in the liver model mice and mitochondrial treated mice.Analysis of GO and KEGG enrichment changes as well as differentially expressed genes revealed that redox and cell cycle related genes and signaling pathways were significantly different after exogenous mitochondrial treatment.Therefore,we speculate that exogenous mitochondria play a role in the treatment of liver fibrosis mainly by reducing oxidative stress damage and inhibiting the proliferation of hepatic stellate cells.