Mitochondrial transplantation therapy inhibit carbon tetrachloride‐induced liver injury through scavenging free radicals and protecting hepatocytes

Abstract Carbon tetrachloride (CCl4)‐induced liver injury is predominantly caused by free radicals, in which mitochondrial function of hepatocytes is impaired, accompanying with the production of ROS and decreased ATP energy supply in animals intoxicated with CCl4. Here we explored a novel therapeutic approach, mitochondrial transplantation therapy, for treating the liver injury. The results showed that mitochondria entered hepatocytes through macropinocytosis pathway, and thereby cell viability was recovered in a concentration‐dependent manner. Mitochondrial therapy could increase ATP supply and reduce free radical damage. In liver injury model of mice, mitochondrial therapy significantly improved liver function and prevented tissue fibrogenesis. Transcriptomic data revealed that mitochondrial unfold protein response (UPRmt), a protective transcriptional response of mitochondria‐to‐nuclear retrograde signaling, would be triggered after mitochondrial administration. Then the anti‐oxidant genes were up‐regulated to scavenge free radicals. The mitochondrial function was rehabilitated through the transcriptional activation of respiratory chain enzyme and mitophage‐associated genes. The protective response re‐balanced the cellular homeostasis, and eventually enhanced stress resistance that is linked to cell survival. The efficacy of mitochondrial transplantation therapy in the animals would suggest a novel approach for treating liver injury caused by toxins.

have become potential therapeutic approaches in rescuing cell function. 5,6 It is well known that mitochondria play crucial roles in bioenergy production and survival of cells. Recovery of mitochondrial function is suggested to restore cell viability, which has become a promising strategy for treating mitochondrial dysfunction related diseases. 7,8 Recently, increasing evidence shows that mitochondria can directly enter cells for medical applications. 9,10 The purpose of the mitochondrial transplantation therapy is to substitute the intracellular dysfunctional mitochondria with healthy exogenous mitochondria to cure mitochondria-associated diseases, which will benefit the treatment of the diseases caused by irreversible mitochondrial impairment. For examples, Cowan et al. reported intracoronary exogenous mitochondrial delivery into the ischemic heart for cardioprotection, 11 and Gollihue et al. stated that healthy exogenous mitochondria could produce functional neuroprotection in the experimental spinal cord damage. 12 In our recent studies, systematic administration of isolated mitochondria retards liver damage initiated by acetaminophen and a high-fat diet. 13,14 The biochemical mechanism of the mitochondria was proved to promote ATP production, as well as powerfully eliminate free radicals. Thus, we assumed that the mitochondrial therapy might recover hepatocytes from free radicals in CCl 4 -induced injury.
In this study, we administrated the isolated mitochondria into the model mice which was intoxicated by CCl 4 , and the therapeutic effect of the mitochondria was evaluated by biochemical measurements and transmission electron microscope (TEM) observation. In order to elucidate the molecular mechanism of mitochondrial therapy, transcriptomics study was executed for the investigation of the differentially expressed genes (DEGs) and associated signaling pathways involving in the recovery of liver function. The research findings will provide a novel insight into the treatment of free radical-induced tissue injury.

| Mitochondria entered cells through macropinocytosis
The liver mitochondria showed good dispersion and exhibited red fluorescence observed by confocal microscope (Figure 1a). Mitochondrial bilayer membrane was intact, and cristae was compact and regularly arranged under TEM (Figure 1b).
Endocytosis is a potential energy-dependent process through which macromolecules enter cells. Here we respectively used inhibitors of clathrin-mediated endocytosis and macropinocytosis, chlorpromazine and amiloride, to investigate the entry pathway of the mitochondria. After chlorpromazine addition into cell media, fluorescence intensity did not show apparent changes compared with control cells (Figure 1c). However, cells treated with amiloride exhibited very weak fluorescence, indicating that the cell internalization of mitochondria was inhibited. Thus, mitochondria entered cells through macropinocytosis pathway in the study.

| Mitochondria prevented CCl 4 -induced cell injury
CCl 4 is an industrial solvent with substantial hepatotoxicity. In hepatocytes, CCl 4 is metabolized to produce trichloromethyl free radical (CCl 3˙) , and further the CCl 3˙r adical treated with oxygen to produce trichloromethyl peroxy free radical species (CCl 3 OO˙). The two species of free radicals can bind to lipids and proteins of biological membranes, leading to hepatocyte impairment. In this study, CCl 4 significantly decreased cell viability (Figure 2a), meanwhile the content of MDA (a lipid peroxides product) increased, and the levels of antioxidants (SOD and GSH) and ATP reduced (Figure 2b-e). However, after the addition of exogenous mitochondria into the hepatocytes cell media, cell viability elevated with the concentration-and timedependent pattern. The levels of anti-oxidants, ATP and mitochondrial ND, increased, while ROS level significantly reduced after mitochondrial treatment (Figure 2f). The results suggested that mitochondria could increase cell viability through increasing ATP production and diminishing free radical.

| Mitochondria distributed in the liver after intravenous injection
After mice were administrated with fluorescence-labeled exogenous mitochondria (0.4 mg/kg body weight) for about 4 h, tissue fluorescence was observed under in vivo imaging, and fluorescence of the tissue sections was recorded by confocal microscope (Figure 3a,b).
The results indicated that the mitochondria through intravenous injection distributed in the liver, lung and kidney, and a small amount in heart. Besides, the livers impaired by CCl 4 exhibited the strongest fluorescence compared to other tissues and control livers, which might be due to the destruction of the liver structure by CCl 4 , resulting in an increase in the amount of mitochondrial entry.

| Mitochondria inhibited CCl 4 -induced chronic liver injury and fibrosis
CCl 4 is widely used to establish animal model of liver injury. The pathological process induced by CCl 4 undergoes acute and chronic injury, subsequently liver fibrosis, and finally cirrhosis. When CCl 4 dissolved in olive oil is administrated to mice for only once, it can induce acute hepatocellular injury. 15 When CCl 4 is repeatedly injected, the impairment becomes aggravating and irreversible, followed by liver fibrosis and cirrhosis.
Here we respectively administrated the mice for 3 and 5 weeks' with CCl 4 injection to induce chronic liver injury and fibrosis model to examine the effect of mitochondria therapy. As shown in Figure 3c To further determine the mitochondrial function, we measured the levels of mitochondrial membrane potential and respiratory chain-related enzymes. The results showed that mitochondrial therapy almost wholly reversed the decreased membrane potential caused by CCl 4 (Figure 4a), and the activities of both SDH and PDH increased (Figure 4c,d).
Moreover, CCl 4 -induced fibrosis is a stable model over a 5-week period following cessation of CCl 4 exposure. We utilized this model of chemically produced fibrosis to evaluate the anti-fibrosis potential of the mitochondria by continuously administering them for 7 days. The healthy liver showed a smooth surface, while it became rough after CCl 4 intoxication (Figure 5a). Mitochondrial therapy improved the liver surface morphology and shrunk the fibrotic area on sections by Sirius red staining (Figure 5b,c). Besides, a biochemical assay of hydroxyproline content indicated that the hydroxyproline, a biomarker of fibrosis, was suppressed in the liver tissues of the model mice after the treatment with the mitochondria (Figure 5d).

| The functional assortment of DEGs
RNA-seq datasheet showed that DEGs with 2.5 or higher folds between mito-treated and control groups were differentiated upon

| Mitochondria activated the anti-oxidant system
CCl 4 is known to produce reactive free radicals and initiation of cell damage through conjugation of the free radicals to the membrane proteins. CCl 4 and the generated ROS are associated with impaired glutathione metabolism, finally counteracting the anti-oxidant effects.
Also, increasing ROS simulate a vital role in the production of the liver injury and initiation of the hepatic fibrogenesis. However, the cellular anti-oxidant system was highly activated by the mitochondrial administration. From the KEGG pathway, glutathione metabolism was significantly up-regulated by mitochondria. In addition, a series of genes of anti-oxidant enzymes, including PDH, GPx, PON, SOD, CAT, and TXNR, was transcriptionally up-regulated (Table S1). The activation of these enzymes states that an enormous quantity of ROS was eliminated from the liver after the mitochondrial treatment.
The result was further identified by biochemical assay, in which ROS level significantly reduced from 412.98 to 241.40 a.u/gprot (mito-low group) and 152.24 a.u/gprot (mito-high group), respectively, while GSH content increased from 5.51 to 8.91 mmol/gprot (mitolow group) or 11.50 mmol/gprot (mito-high group) after the mice F I G U R E 4 Biochemical measurement of mitochondrial activities in mouse livers after mitochondrial treatment. (a) JC-1 assay. Also, activities of mitochondrial ND (b) PDH (c) and SDH (d) were respectively measured after mitochondria were administered into the mice. Data were expressed as mean ± SD of the mean (n = 10 mice for each group). ## p < 0.01 compared with the control; *p < 0.05, **p < 0.01 comparison with the CCl 4 group were donated with the exogenous mitochondria (Figure 7a

| Mitochondria restored oxidative phosphorylation function
CCl 4 can impair the respiratory chain of liver mitochondria and induce the marked respiratory inhibition that leads to a decrease of ATP generation. However, a series of oxidative phosphorylation (OXPHOS)related genes, including Coq10a, Ndufaf1, Fmc1, Ndufs7, Sdhb, Mrpl9, and Atp5d, are up-regulated to rebuild the respiratory chain and ATP synthase (Table S2). Notably, the genes of mitochondria-coded ATP synthase 6 (Mt-ATP6) and COX2 subunit (mt-Nd2) were also activated.
The up-regulated OXPHOS-associated genes would result in the increases of ND activity and ATP production after mitochondria treatment, which was identified by the biochemical measurements

| Mitochondria prevented cell proliferation
The biology of acute liver injury is characterized by hepatocellular damage and inflammation. Thus, cell cycle arrest represent a vital role in the prevention of hepatocytes and maladaptive repair following hepatocellular injury, avoiding replication of damaged DNA and carcinogenesis. 20 In this study, an extensive range of genes associated in the cell proliferation were recognized to be considerably inhibited by the mitochondria and most of them belonged to the ZH-C2H2 transcription factor-mediated cascade system (Table S4) Moreover, the levels of (e) ATP content and (f) ND activity were determined, respectively. Each group contained eight mice (n = 8 for each group). ## p < 0.01 compared with the control; *p < 0.05, **p < 0.01 compared with the CCl 4 group representative cell cycle regulators, cyclin A and B, were significantly down-regulated, evaluated by WB analysis (Figure 8a,e,f). The cell cycle arrest induced by the cascade system will prevent the gathering of DNA mutations that propagate into cancer.

| Mitochondria accelerated the xenobiotic metabolism transformation
Since CCl 4 and its metabolites can induce hepatocyte injury, drugs that accelerated the xenobiotic metabolism would protect the cells.

| Mitochondria maintained liver protein homeostasis
After hepatocyte damage occurred, globulin production in the liver will decrease, and protein homeostasis (proteostasis) will be damaged.

| Molecular signal transduction extrapolation
In order to frame out the all relevant, effective cell signaling pathways produced by the mitochondria, we outlined the gene regulatory network frame through mapping of the RNA-seq data and the existing DNA-protein interaction database and protein-protein interplay (Cytoscape 3.5.0 software). Mitochondrial unfold protein reaction (UPR mt )-induced gene transcription was recognized to be the most pertinent factor in a sequence of biological activities from anti-oxidants, OXPHOS, xenobiotic biotransformation, proteostasis, and autophagy. The extrapolation was supported by the up-regulation of gene transcription of UPR mt molecular markers, including Dnaj, HSPs, lonp1, Clpp, atf5 (Table S7). Also, the levels of two representative proteins of UPR mt , activating transcription factor 5 (ATF-5) and heat shock protein 60 (HSP60), were respectively determined by WB, and the result identified that the protein levels elevated after mitochondrial administration (Figure 9a-c). Collectively, we assume that mitochondria may be attacked by CCl 4 after entering liver cells, which would cause mitochondrial stress, and then induce cell cycle arrest and meanwhile promote the UPR mt protective mechanism (Figure 9d).
Further resulting in improving the activity of anti-oxidant enzymes to diminish ROS, increasing autophagy to eliminate damaged mitochondria and organelles, and enhancing OXPHOS to increase ATP production. The signal pathways could explain the molecular mechanism of mitochondria on cell protection. Mitochondria are the organelles of micro-nano size (0.5-1 μm). It is known that macropinocytosis and cathrin-mediated endocytosis may be the entry pathway of nanocapsules in micro-nano size. To clarify the cell entry mechanism of mitochondria, we used the inhibitors to investigate the internalization pathway of the mitochondria.

| DISCUSSION
The result showed that macropinocytosis could be the plausible entry pathway because macropinocytosis inhibitor amiloride inhibits the mitochondrial internalization. The result is consistent with the report that DsRed-and GFP-tagged mitochondria cannot enter cells with amiloride-treated cells. 21 After the mitochondria enter cells, they are transported to lysosomes. Majority of them escape from the lysosomes and play roles in F I G U R E 9 Molecular signal pathways involved in mitochondrial therapy on CCl 4 induced liver injury. (a) WB bonds of ATF5 and HSP60. (b and c) Respective ratio of the gray value of ATF-5 and HSP60 to β-actin. (d) The mechanism of mitochondrial therapy. Mitochondria stress was induced once entering cells, and then activated the UPR mt pathway, a mitochondria-nuclear retrograde signal, to promote gene transcription and expression of a series of protective proteins and enzymes, including anti-oxidant enzyme, OXPHOS-related proteins, biotransformation enzymes, proteostasis, and autophagy-related proteins cytosol. 22 Then the mitochondria exhibit the ability to increase ATP production, activate the anti-oxidative system and reduce the ROS level, which leads to cell viability increase and functional recovery in a certain concentration range. 23,24 Recent reports also suggest that mitochondrial transplantation therapy can lead to enhanced bioenergetics in normal cardiomyocytes, and meanwhile there is no increase in superoxide production. 25 However, only ATP administration does not show improvement in cardiac function, which implies that mitochondrial therapy could induce tissue regeneration. 26 This advantageous ability of the mitochondria has been utilized in the treatment of mitochondria-related diseases, including myocardial ischemia, cerebral ischemia, schizophrenia, depression, aging, and tumor. 21,27 In the study, the elevated serum transaminase activities, and the decreased GSH and SOD levels that is intoxicated with CCl 4, were predominately stopped by the exogenous mitochondrial administration. Also, the mitochondria prevented liver fibrosis and retained the ultracellular structure of the hepatocytes.
Since molecular signal mechanism of mitochondrial therapy is still unclear, we used transcriptomic analysis to address the issue in the study. The results suggest that the mechanism of the mitochondrial therapy on CCl 4 -induced hepatocyte damage is closely associated with the UPR mt -regulated pathway, a protective transcriptional response mediated by mitochondria-to-nuclear retrograde signaling. 28,29 UPR mt can be activated by mitochondrial stress that is induced by mitochondria unfold proteins, pathogens, and toxins, 30 then significantly increased expression of a broad of UPR mt -regulated genes, including HSPs, respiratory chain proteins, homeostasis proteins (proteostasis), detoxification response (ROS defense and toxin detoxification), autophagy, protease, and metabolism-associated proteins. [31][32][33] Activation of the UPR mt promotes the anti-oxidant gene expressions to stabilize mitochondrial function and increase adaptation in mitochondrial stress, which are linked to healthy lifespan and longevity. At present, UPR mt is recognized as a protective mechanism that allows the synchronization of nuclear and mitochondria to maintain cell homeostasis. 34,35 Among the UPR mt -regulated genes, anti-oxidant genes containing PON, SOD, and genes involved in GSH metabolism is activated to prevent the protein and membrane injury induced by free radicals. 36 In this study, the elevated anti-oxidant system will stabilize the intracellular environment and recover functions of salvageable organelles damaged by CCl 4 , and the irreparable organelles could be eliminated via autophagy activated by UPR mt . In addition, UPR mt can up-regulate the expression of xenobiotic detoxification genes such as UGTs and GST, which would accelerate CCl 4 excretion from the body to prevent its hepatotoxicity. CCl 4 is a commonly used toxin to cause liver injury since it can induce free radicals and trigger peroxide chain reaction, leading to lipid peroxidation in phospholipid-rich and membrane structures. Mitochondria contain phospholipid bilayers, and especially the inner membrane is the main site of cellular energy production (OXPHOS). 5 The free radicals damage the capability of OXPHOS and decrease ATP production.
Moreover, free radicals can impair proteins and nucleic acids, and then aggravate cell injury. Thus, CCl 4 -induced liver injury could be prevented through scavenging free radicals and restoring cellular energy supply. 37,38 Here we used the mitochondrial transplantation therapy to deliver healthy mitochondria into mice. The exogenous mitochondria have intact membrane structures, which can recover energy supply.
Also, the mitochondria can activate cell protective UPR mt effect, then promote anti-oxidant system to scavenge free radicals. In addition, the activated autophagy could eliminate the damaged mitochondria and other organelles. Therefore, administration of healthy mitochondria improve hepatocyte function that impaired by CCl 4 .
Nevertheless, the mechanism of the mitochondrial therapy on cell cycle arrest is still unknown. Increasing evidence shows that mitochondrial function is closely associated with cell cycle. 39,40 One of evidence comes from the study of yeast mitochondrial inheritance. 41 The OXPHOS capability of Saccharomyces cerevisiae decreases when the cells enter into mitosis, while the capability is recovered during meiosis, and then decreased again at the post-tetrad stage before budding, implying that increase of OXPHOS might induce cell cycle arrest. A recent report shows that activation of transcription factor daf-16

(FOXO orthologue in mammals) after the UPR mt is activated in
Caenorhabditis elegans, in which FOXO is an important cell regulator to induce cell cycle arrest and DNA repair. 32,42 During liver injury caused by CCl 4 , cell cycle arrest in hepatocytes and nonparenchymal cells would benefit the cell repair from injury at the resting phase, and prevent the progression of liver fibrosis. 43,44 Here we used intact mitochondria to study their function on CCl 4induced liver injury through the intravenous administration. After the mitochondria are injected into blood vessels, they would increase the content of mitochondria in blood. It is known that blood contains intact cell-free mitochondria in both healthy people and patients, which would be important in maintaining normal physiological functions. 45,46 At present, a new concept has arisen recognizing that extracellular mitochondria are important players in tissue regeneration and immune regulation, 47 which will benefit the recovery of liver function that damaged by CCl 4 . Nevertheless, the effects of intact mitochondria on liver cells (including hepatic stellate cells and kuffer cells) and blood immune cells will continue to be further investigated.

| CONCLUSIONS
Today mitochondria are regarded as more than an energy plant in cells. Mitochondria are signaling organelle that maintain cell homeostasis and elicit tissue repair and regeneration. The mitochondrial therapy with protective purposes have made rapid progress from cell and animal studies to clinical trials. 48 Isolated mitochondria can enter cells after simply co-incubation, then play beneficial roles through increasing bioenergy supply and diminishing free radicals. Mitochondrial therapy also prevents liver cell injury in animals with CCl 4 intoxication by activating the signal transduction to nuclear and induce various gene transcription and expression. The protective response reconstructs the stable balance of mitochondria, and intimately enhance stress resistance that is linked to cell survival. The study provides a novel approach for preventing liver injury caused by toxins and suggests that activation of mitochondria-unclear signal pathway would be the molecular mechanism the mitochondrial therapy.

| Mitochondrial isolation and staining
Liver mitochondria from healthy mice were isolated and activity determination according to the earlier reports. 14,24,49 In brief, the mice were euthanatized through quick cervical dislocation. Further, the mouse liver was removed and put in ice-cold PBS (pH 7.4). The liver

| Cell culture
Mouse hepatocytes were cultured according to our earlier report (Shi et al. 14 ). The cells were preserved in Dulbecco's modified Eagle's medium (DMEM) complemented with 10% FBS at constant temperature and sterile culture incubator along with 5% CO 2 at 37 C (ESCO, Indonesia). All media were obtained from Gibco.

| Cell internalization of mitochondria
To elucidate the mechanism of cell entry of the isolated mitochondria, macropinocytosis inhibitor amiloride and cathrin-mediated endocytosis inhibitor chlorpromazine, were respectively put on into the cell media. After 30 min, mitochondria were introduced into the DMEM and further incubated for another 4 h. The cells were further cleaned using PBS for 3 times. Nucleus was stained by DAPI. Fluorescence was observed, and images were obtained with a confocal microscope (Zeiss LSM 510, Germany).

| CCl 4 -induced cell injury and biochemical assay
When the cells grew up, CCl 4 (10 mM) were introduced inside cul-

| Biochemical assay and tissue evaluations
Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were respectively estimated by standard procedures of automatic biochemistry analyzer. Hydroxyproline content was estimated using the Jamall methods as earlier reported.
UDP-glucuronosyltransferase (UGT) level was measured according to the commercial kit (Shanghai Jianglai industrial Co., Ltd., China). Moreover, liver tissues were settled in the 10% buffered formalin and sectioned on a paraffin microtome. Then the sections of the liver tissues were stained using hematoxylin-eosin (HE) or Sirius red staining.

| Liver mitochondrial activity assay
To measure mitochondrial activity in vivo after mitochondrial treatment. Mitochondria from mouse liver were extracted 24 h after the exogenous mitochondrial injection. The activities of the extracted mitochondria were respectively measured. Also, TEM was used to observe the mitochondrial morphology.

| Western blot analysis
Protein extraction of liver specimens was exposed to SDS-PAGE and transmitted onto PVDF membrane, further investigated with antibodies versus LC3B, parkin, cyclin A and B, ATF-5, HSP60 and β-actin (Beijing boason Biotech. Co., Ltd., Beijing, China), then with HRP-conjugated secondary antibodies (1:5000; Beijing Dingguo Biotech. Co., Ltd., Beijing, China) as the secondary antibody. After the membranes were washed twice for about 15 min each using wash buffer, the signal was identified by the ECL system (Pierce Co.).

| RNA extraction, library preparation, and Illumina Hiseq4000 sequencing
The whole RNA of mouse liver (100 μg/ml) was acquired utilizing TRI Reagent ® based on the manufacturer's procedure (Sigma-Aldrich, Co.). The quality of the RNA was estimated utilizing the Agilent 2100 Bioanalyser system (Agilent, USA) and quantified utilizing the

| Read mapping and sequence assembly
The untreated raw and pure paired-end reads of the sequence were cut, trimmed, and the sequence quality was controlled by SeqPrep and Sickle with revert existing parameters. Further, the alignment of pure reads was uniquely maintained based on the reported genome reference utilizing orientation mode with Bowtie2 software. Further particular zone of the gene was expanded according to subsequent depths of sites, and then the operon was acquired. In supplement to this, further, the whole genome of RNA was further split into numerous fragments of 15 kb windows, which serve 5 kb each. The newly transcribed zone was stipulated as additional two successive windows without the overlapped zone of the gene, where a minimum two reads where mapped per window in the identical orientation.

| Differential expression analysis and functional enrichment
Further, for recognizing the differential expression genes (DEGs) among the two types of the sample groups, the level of expression of the genes for each and every transcript was determined to utilize the fragments per kilobase of exon per million mapped reads (FRKM) methodology. Cuffdiff was utilized to study the primary differential gene expression, and the examination of DE-seq is conducted through R software was then initiated to test numerous DEGs. The DEGs among exogenous mitochondrial administration therapy in the sample groups and control groups were recognized utilizing the upcoming criteria: (1) the logarithmic value of fold change should be higher than 2.5, (2) the FDR range should be lower than 0.05. Further, to estimate the function of the DEGs, gene ontology (GO) functional enhancement and KEGG pathway examination were performed by Goatools and KOBAS tools. DEGs were predominantly enhanced in GO terms and cellular metabolic pathways when Bonferroni-corrected p < 0.05.

| Statistical analysis
The collected data were demonstrated as mean ± SD for every sample. Numerous comparisons among the control groups and other groups were examined by Dunnett's test. Differences were observed considerable when p < 0.05.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.