**4. Discussion**

In this study, we demonstrated that mitochondrial dysfunction in hepatocytes is closely linked to hepatic fibrosis, and HtrA2/Omi might play a critical role in preventing of hepatic fibrogenesis through regulating mitochondrial homeostasis. Mitochondria are vital intracellular organelles that are altered in response to cellular stress and metabolic changes in hepatocytes. Oxidative stress is considered a key accelerator of liver fibrosis, and ROS produced by hepatocytes promote HSCs activation, resulting in excessive ECM deposition [30,31]. Furthermore, increased ROS production is associated with mitochondrial dysfunction in hepatocytes during liver damage [23,32], and subsequent mitochondrial dysfunction leads to oxidative stress and changes in mtDNA damage and calcium homeostasis, resulting in an energy crisis that can eventually lead to hepatocyte death. In the present study, we evaluated the alteration in the mitochondrial structure or function in a mouse model of CCl4-induced liver fibrosis. In addition, we demonstrate that downregulation of HtrA2/Omi expression in CCl4-induced liver fibrosis has a major role in modulating mitochondrial function and ROS generation in vivo and in vitro. Studies have shown that mitochondrial dysfunctions associated with HtrA2/Omi is a key causative factor inducing cell death in various chronic pathological conditions in numerous human diseases, such as neurodegeneration and cardiovascular diseases [33,34]. However, the relationship between mitochondrial dysfunction induced by HtrA2/Omi and chronic liver disease is unclear. Our analysis revealed that HtrA2/Omi-deficient and HtrA2/Omi-mutant hepatocytes have considerable reductions in mitochondrial electron transport chain activity and altered mitochondrial ultrastructural organization. However, we found that mitochondrial mass remained unchanged and the production of total ROS and mtROS increased in HtrA2/Omi deficient hepatocytes. These observations are consistent with previous studies demonstrating that HtrA2/Omi deficiency is involved in the accumulation of intracellular ROS [12,35,36], suggesting that mitochondrial damage in chronic liver injury leads to oxidative stress and changes in mtDNA damage that can eventually result in hepatocyte death. Although there was no change in the mitochondrial mass, the reason for the increased mtDNA contents in HtrA2/Omi deficient or mutation hepatocytes was that the destruction of mtDNA, which exists as a supercoiled form, caused morphological transfer to mtDNA of relaxed circular and linear forms. Accumulating evidence has demonstrated an association that the accumulation of relaxed and linearized mtDNA was used as a relatively good template for DNA amplification.

A number of recent studies reported correlations between elevated levels of intracellular or circulating mtDNA and various human diseases [37–39]. A study by Zhang et al. found that traumatic injury induces the release of mitochondrial DAMPs such as formyl peptides and mtDNA into the circulation, and these circulating mitochondrial DAMPs activate multiple inflammatory signal pathways through a specific receptor, toll-like receptor 9 (TLR9) [40]. TLR9 is in intracellular compartments and recognizes unmethylated cytosine phosphate guanine-containing DNA. Similarly, serum mtDNA levels are significantly higher in patients where acetaminophen-induced liver injury is significantly higher than in healthy controls, suggesting that the extent of mtDNA release into the circulation can be measured as a mechanistic biomarker of mitochondrial damage in patients with liver injury [41–43]. However, studies on how damaged mtDNA accumulated in hepatocytes or mtDNA released from hepatocytes a ffect non-parenchymal liver cells such as HSCs and cause liver fibrogenesis are still limited. Although we did not extract the damaged mtDNA released from HtrA2 /Omi deficient and HtrA2/Omi mutated hepatocytes, it showed the possibility that the accumulation of damaged mtDNA in hepatocytes can induce HSC activation in the form of DAMP molecules. These results were the first to demonstrate that HtrA2/Omi expression regulates mtDNA damage and mitochondrial homeostasis in hepatocytes during liver fibrogenesis.

Initially, the roles of HtrA2/Omi in apoptosis, mitochondrial protein folding quality control, and cell survival were investigated [9]. A recent study reported that HtrA2/Omi regulates autophagy and inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC [44]. In addition, Michell et al. found that expression of the pro-apoptotic protein Bcl-2 in the liver protected against CCl4 induced-mitochondrial dysfunction and -oxidative stress in hepatocytes [8]. Therefore, we observed that, by protecting the mitochondria, HtrA2/Omi could restore mitochondrial structure and mitochondrial function, and subsequent play a pathophysiological role in the liver fibrogenesis in CCl4-induced liver fibrosis model. By hydrodynamics-based gene transfer, the overexpression of HtrA2/Omi leads to antifibrotic e ffects in CCl4-induced liver fibrosis mice model through decreasing collagen accumulation and enhancing anti-oxidative activity by modulating mitochondrial homeostasis in the liver. These results sugges<sup>t</sup> that suppression of HtrA2/Omi expression promotes hepatic fibrogenesis by modulating mitochondrial ROS generation, and these novel mechanistic insights involving the regulation of mitochondrial homeostasis by HtrA2/Omi may be of importance for developing new therapeutic strategies for hepatic fibrosis.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/10/1119/s1. **Figure S1.** Lentivirus-mediated HtrA2/Omi depletion in hepatocyte lead to impaired mitochondrial function and metabolism. ( **A**) HtrA2-shRNA encoding lentivirus were infected at MOI of 5 into FL83B cells. HtrA2/Omi protein levels were assayed using western blotting in lenti-shHtrA2 and lenti-shNC hepatocyte. (**B**) Mitochondrial morphology was measured by FACS-analysis using MitoTracker green staining. Western blotting for VDAC, mitochondrial mass proteins in lenti-shHtrA2 and lenti-shNC hepatocyte, with β-actin as a loading control. (**C**) Exemplary flow cytometry plot and histogram of showing populations of primary hepatocyte in light scatters; FSC and SSC. The fluorescence mean intensity of FSC and SSC was quantified. **Figure S2.** Loss of HtrA2/Omi in hepatocyte results in the accumulation of dysfunctional mitochondria and oxidative stress. (**A**) Changes of mitochondrial ultrastructure in liver tissue from WT or mnd2-mutant mice at postnatal day 40. Appearance of normal (WT, left panel), mnd2-mutant (right panel) mice and liver. (**B**) H&E staining (original magnification, X200) and TEM images of liver sections. (**C**) Representative TEM images of hepatocytes isolated from WT and mnd2-mutant mice. (**D**) qRT-PCR analysis of mt-ND1 and mt-COX1 DNA normalized to nuclear Ndufv1 DNA in primary hepatocyte. (E & G) ND1, COX1, ATP5A, COX5B and ERRα mRNA expression in hepatocyte, as determined by qRT-PCR. (F) Damaged mtDNA levels of the 79 bp fragment (damaged) and 230 bp fragment (undamaged) in gDNA isolated from hepatocyte of mice were assessed by qPCR. Bars represent mean copy number ratios of mtDNA-79:230, normalized to 18S levels. **Figure S3.** Loss of HtrA2/Omi mitochondrial protease activity promotes liver fibrosis. (**A**) Loss of serine protease activity of HtrA2/Omi in liver sections from WT and mnd2 heterozygous (mnd2/+) mice. Liver lysates were immunoprecipitated (IP) with HtrA2/Omi-specific polyclonal antibody. The IP complexes were incubated for the indicated times at 37 ◦C with β-casein as a substrate. The reaction samples were resolved by 15% SDS-PAGE, and the processing pattern of β-casein was visualized by staining with Coomassie Brilliant Blue dye (CBB). The level of the HtrA2 was analyzed by IB with HtrA2/Omi Ab. (**B**) Representative images of H&E and Sirius red staining of liver sections from WT and mnd2 heterozygous (mnd2/+) mice via repetitive CCl4 injections (original magnification, X200). (C) Semi-quantitative analysis of Sirius red staining in the fibrotic livers. (**D**). The hydroxyproline content. Figure S4. HtrA2/Omi expression in mouse model of CCl4-induced liver fibrosis alleviates liver fibrosis by protecting hepatocytes damage. HtrA2/Omi expression in CCl4-treated mice following the hydrodynamic tail vein injection of HtrA2/Omi-encoding plasmid DNA (*n* = 7) compared with the mock control group (*n* = 5) over 8 weeks. Hepatic fibrosis was induced by injection of CCl4 two times per week for 8 weeks. (A&B) HtrA2/Omi expression alleviates CCl4-induced hepatocyte death. Representative pictures of TUNEL and activated caspase 3 staining for apoptosis detection. Nuclei were stained with DAPI (blue). (C) The immunohistochemistry staining for cleaved caspase-3 was quantified by Image-Pro Plus 6 software. (D) ATP5A and COX5B mRNA expression in liver tissue, as determined by qRT-PCR.

**Author Contributions:** B.Y.K., S.M.K., and G.W.L. conducted experiments. W.H., J.-H.K., M.-K.N., and H.R. analyzed data and interpreted the results. W.H. and S.K.Y. designed the studies and wrote the manuscript. All coauthors reviewed the manuscript.

**Funding:** This research was supported by grants of the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2016R1D1A1B03931395 & 2017R1D1A1A09000911 & 2019R1I1A1A01056842) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (HI17C0509).

**Conflicts of Interest:** The authors declare no conflict of interest.
