Liver fibrosis is the result of long-term stimulation of the liver by damaging factors and will eventually evolve into cirrhosis. With the advanced understanding of the molecular mechanism of liver fibrosis, it is gradually recognized that liver fibrosis is a reversible process. In 1995, the National Institutes of Health (NIH), after summarizing the past experience of gene therapy, pointed out that the development of vector technology, the study of molecular mechanisms of disease and the development of animal models should be the right direction for gene therapy research. This paper focuses on the research of molecular mechanism of liver fibrosis and the application of vector technology in the gene therapy of liver fibrosis. 1. Gene therapy research on molecular mechanisms of liver fibrosis 1.1 Regulation of hepatic stellate cells Activation and proliferation of hepatic stellate cells (HSC) are the central link and common pathway for the development of liver fibrosis. Activated HSC express α-smooth muscle-alpha (α-SMA) and glial fibrillary acidic protein (GFAP). Activated HSC can secrete various extracellular matrix (ECM) components of the fibrotic process, such as type I, III, IV collagen and laminin, and activated HSC can also secrete matrix metalloproteinase (MMP) and its inhibitor matrix metalloproteinase tissue inhibitors The activated HSC can also secrete matrix metalloproteinase (MMP) and its inhibitor, tissue inhibitors of metalloproteinases (TIMP), causing a relative lack of ECM degradation and excessive deposition in the liver, leading to the formation of liver fibrosis. Therefore, the regulation of HSC activation and proliferation is an important way of gene therapy for liver fibrosis. 1.1.1 Inhibition of HSC activation Since oxidative stress and inflammatory response in the liver are the initiating links in the development of liver fibrosis, many inflammatory factors can activate HSC and lead to the development of liver fibrosis. Antioxidants such as Silybum, phosphatidylcholine derived from legumes, and S-adenosyl-L-methionine have been found to reduce the extent of liver fibrosis by inhibiting the activation of HSC. Chen et al. demonstrated that IFN-α effectively inhibited HSC activation and reduced the expression of α-SMA and mRNA levels of TIMP-1 and transforming growth factor (TGF-β) in HSC cells. Some other cytokines such as tretinoin A, glycyrrhetinic acid , retinyl palmitate and resveratrol have also been shown to inhibit HSC activation. TGF–β is the most effective stimulating factor for HSC activation. During liver injury, ECM releases TGF-β, which can promote HSC activation but inhibit HSC proliferation, as well as inhibit hepatocyte proliferation and induce hepatocyte apoptosis; TGF-β promotes collagen synthesis through Smad signaling pathway, inhibits MMP synthesis and promotes TIMP synthesis; and the reduction of MMP synthesis will lead to insufficient ECM degradation, resulting in ECM accumulation. Some anti-TGF-β receptors have obtained good results in animal experimental models; serine protease inhibitors can block the activation of TGF-β; Smad-7 transfer factor can block the intracellular signaling pathway of TGF-β; our study also confirmed that the activation of TGF-β on HSC was inhibited by RNA interference with the expression of Smad-2 on the TGF-β transduction pathway . Peroxisome proliferator activator receptor gamma (PPAR-γ) can control cell growth and differentiation, and thiazolidinediones as PPAR-γ ligands can inhibit HSC activation and promote fibrinolysis. Captopril (angiotensin-converting enzyme inhibitor) and candesartan (angiotensin II receptor antagonist) can also reduce the extent of liver fibrosis by acting on HSC and promoting fibrinolysis. 1.1.2 Inhibition of HSC proliferation Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor, insulin-like growth factor (IGF-I) can promote HSC proliferation through tyrosine kinase receptor signaling pathways in acute and chronic liver injury. Among them, PDGF is particularly important, and the phosphodiesterase inhibitor 3-7-dimethylxanthine can block PDGF-related HSC mitosis. The anti-angiogenic properties of trinitrophenyl (TNP-470) also inhibit HSC proliferation. And small molecule tyrosine kinase receptor inhibitors are expected to be used in liver fibrosis treatment by further modification. 1.1.3 Induction of HSC apoptosis The spontaneous reversal of hepatic fibrosis is mainly dependent on HSC apoptosis, which reduces the number of HSC apoptosis, decreases ECM secretion, decreases TIMPs synthesis, increases MMPs activity, and increases ECM degradation, thus promoting the reversal of hepatic fibrosis. The death receptor pathway, mitochondrial pathway and endoplasmic reticulum pathway are the most important pathways mediating HSC apoptosis. HSC express many cell surface death receptors, such as Fas ligand, TNF-α, nerve growth factor (NGF), etc., which can induce HSC apoptosis in vitro by acting on the corresponding receptors; diazepam promotes HSC apoptosis by acting on the peripheral benzodiazepine receptor (PBR) of the mitochondrial pore; branched mycoplasma and salazosulfamerazine promote HSC apoptosis by inhibiting nuclear transcription factor (NF-κB) to promote HSC apoptosis. 1.2 Promotion of extracellular matrix degradation The imbalance between synthesis and degradation of ECM, matrix remodeling and abnormally increased deposition of interstitial collagen component type I/III collagen are the pathological basis of liver fibrosis. Therefore, increasing the gene expression of collagenases, regulating the biological activity of endogenous collagenases and degrading the increased ECM are the keys to reversing the process of liver fibrosis. MMPs are a group of proteolytic enzymes that degrade ECM and play a key role in the process of ECM degradation, and the activity of MMPs is closely related to the expression of their specific inhibitors, TIMPs, in tissues. Currently, MMPs and TIMPs have become important targets for gene therapy of liver fibrosis, and the MMPs family consists of four main categories, namely interstitial collagenases, gelatinases, interstitial lysins, and membrane-type MMPs (MT-MMPs), depending on the substrate. The identified TIMPs include TIMP-1, -2, -3 and -4, all of which can bind to specific active MMPs to form 1:1 complexes and inhibit the degradation activity of the latter on ECM. It was found that transfection of MMPs expression plasmids or antisense oligonucleotide plasmids of TIMPs in hepatic fibrosis rats promoted the degradation of type I/III collagen and reversed fibrosis; reproductive hormone relaxin (relaxin) also reduced collagen deposition due to HSC activation by downregulating TIMPs; proline-4-hydroxylase inhibitors could relatively increase the activity of MMPs, thereby promoting ECM degradation and reversing liver fibrosis. Urokinase plasminogen activator (uPA) is the initiator of the ECM cleavage protein cascade. uPA also increases the expression of hepatocyte growth factor (HGF) and upregulates the activity of MMPs, promoting the reversal of hepatic fibrosis and hepatocyte regeneration. 2.The application of vector technology in gene therapy of liver fibrosis Effective gene transfer is the key to gene therapy, and it is extremely important to choose a suitable vector to maximize the effectiveness of cytokines in target cells. The ideal vector should have the advantages of high specificity, high affinity, large gene carrying capacity, high efficiency of integrated transfection, low antigenicity and toxicity, and long duration of expression of functional genes. Gene vectors are divided into two categories: viral vectors and non-viral vectors. Non-viral vectors include liposome-encapsulated DNA, multimers (poly-lysine complexes, etc.) and recombinant celiac remnants, etc. Non-viral vectors have the advantages of large gene carrying capacity, less antigenicity and less toxicity, but at the same time, they have the disadvantages of low efficiency and short duration of functional gene expression, therefore, viral vectors account for more than 70% of all vectors currently studied and used. 2.1 Viral vectors Currently, viral vectors are considered to be the most effective tools for gene transfer, and retrovirus, adenovirus and adeno associated virus (AAV) are the most promising vectors for gene therapy of liver fibrosis. The adenovirus vector is a double-stranded DNA molecule, which determines the most important characteristic of its physicochemical stability. Adenoviral vectors can infect both dividing and non-dividing cells, and the expression of target gene products delivered by adenovirus is high enough to reach therapeutic levels. In view of these advantages, adenoviral vectors have been widely used in various aspects of gene therapy for hepatic fibrosis. qi et al. constructed a replication-deficient adenoviral vector expressing the extracellular region of TGF-β type II receptor to block the endogenous TGF-β receptor signaling pathway; Rederfeld et al. introduced mutants of MMP-9 into CCl4-induced BALB/c mice to bind TIMP-1 for the purpose of knocking down TIMP-1; Salgado et al. constructed defective adenovirus-mediated non-secretory human uPA gene to induce collagenase expression. However, adenoviral vectors have the disadvantage of short duration of expression of the target gene, and because adenoviruses are immunogenic, they tend to cause immune reactions and toxic side effects in the host. AAV is a single-stranded non-pathogenic DNA virus, which can be integrated into chromosome 19 in a targeted manner, and with the assistance of a helper virus, AAV can carry out virulence-producing infections. AAV-mediated human IFN-γ (hIFN-γ ) expression vector was constructed by Chen et al. to inhibit the activation of HSC; Tsui et al. injected AAV-mediated type I subtilisin heme oxygenase gene (HO-1) into liver fibrosis animal model through portal vein and obtained satisfactory transfection efficiency and stable expression. Retroviruses are enveloped RNA viruses and can be divided into two categories according to their genome structure: simple retroviruses such as mouse leukemia virus (MLV) and more complex retroviruses such as human immunodeficiency virus (HIV) in the genus lentivirus (LV), HIV). Simple retroviruses were first used in gene therapy for liver fibrosis. Because it can only transfer exogenous genes to proliferating and dividing cells, and has poor transcriptional effect on resting cells, previous scholars had to use damaging means such as partial hepatectomy to induce hepatocyte division, and the disadvantages such as poor stability of viral particles, small amount of accommodating exogenous genes, and susceptibility to rapid destruction by complement limited his use. Lentiviruses are members of the family Retroviridae. The main lentiviruses isolated so far are: HIV, eqine infectious anemia virus (EIAV) and simian immunodeficiency virus (SIV). Unlike other retroviruses, lentiviral genomes are complex, containing three structural genes, including gag, pol, and env, and 5′ and 3′ end LTR structures, as well as four accessory genes, vif, vpr, nef, vpu, and two regulatory genes, tat and rev. The infected or transformed animal cells can be transmitted continuously, so the lentivirus can be used as a vector to change the genotype of animal cells and inherit it to the offspring. The most important feature of lentiviral vectors is that they can infect dividing cells as well as non-dividing cells, and can be expressed in the host for a long time, and they are less likely to induce host immune responses and have good safety. In addition, the lentivirus is suitable for multiple gene expression systems because of its powerful packaging capacity, which is generally considered to be up to 8-10 kb. Several experiments in animal models have shown that hepatocytes are susceptible to LV-mediated gene transfer and that the transferred exogenous genes can be expressed for a long time. zahler et al. found that the infection efficiency of LV can reach 30-40% in primary cultured fetal hepatocytes. lv was modified to efficiently transduce primary cultured rat hepatocytes and primary cultured human hepatocytes in vitro. We have obtained stable expression by constructing a lentivirus-mediated tTG gene RNA interference plasmid transfected with rat primary HSC. Based on the good properties in hepatocyte gene transduction, the application of lentiviral vectors in liver fibrosis gene therapy has attracted more and more attention, and their safety has been gradually ensured in continuous improvement. 2.2 Targeting of vectors The selection of liver-specific targeting vectors is gradually gaining attention, as there may also be targets in extrahepatic tissues and organs, so the emphasis on the specificity and targeting of therapeutic effects can not only improve the efficiency of the effect but also reduce the potential toxic effects on other organs. Inagaki et al. found that by adding the enhancer sequence COL1A2 to the vector promoter, the exogenous gene was only expressed in the liver fibrosis group of mice with the specific enhancer. The exogenous gene was expressed only in activated HSC of mice with liver fibrosis, but not in the liver and other organs of normal mice. Therefore, further screening of highly selective targeting vectors against liver parenchyma and mesenchymal cells for liver fibrosis gene therapy will improve the targeting and efficiency of liver fibrosis treatment and enhance the overall level of anti-liver fibrosis treatment. In conclusion, with the deepening understanding of the molecular mechanism of liver fibrosis, gene therapy for liver fibrosis has shown good prospects for development; similarly, the development of vector technology has also provided a guarantee for improving the targeting, safety and efficacy of gene therapy for liver fibrosis. However, liver fibrosis is a result of multi-gene and multi-pathway regulation, and most gene therapies are still limited to animal experimental stage. Therefore, there is a long way to go in the research of gene therapy for liver fibrosis, and the use of multi-linked combined gene therapy and further improvement of the targeting and safety of vector systems are important directions for the development of gene therapy for liver fibrosis.