Melatonin pretreatment can improve the therapeutic effect of adipose-derived stem cells on CCl4-induced liver fibrosis

Abstract In this study, the therapeutic effect of Mel-incubated Adipose-derived mesenchymal stem cells (ADSCs) on CCl4-induced hepatic fibrosis was investigated. Mice with hepatic fibrosis were intraperitoneally injected with 8% CCl4 twice a week for 4 weeks. Starting from week 5, mice in the ADSC group and ADSC + Mel group were injected with 1 mL PBS cell suspension containing 1 × 106 ADSCs or ADSCs pretreated with 50 nM Mel twice a week for 2 consecutive weeks. Results: In the model group, severe histopathological changes were observed in the liver, including severe vacuolation, nuclear fragmentation, and liver fibrosis, and these changes were ameliorated by Mel-pretreated ADSCs. RT-qPCR results showed that Mel-induced ADSCs significantly inhibited the expression of proapoptotic genes (Caspase-8, Bax, and Caspase-3) and promoted the expression of an anti-apoptotic gene (Bcl-2). Immunohistochemical results showed that a large number of MMP-9-, TGF-β-, and MMP-2-positive cells were present in the liver tissues of the model group, while the number of positive cells was reduced by Mel-induced ADSCs. ELISA results showed that the ADSC pretreated with Mel also significantly reduced the expression levels of TNF-α and IL-6. Conclusion: ADSCs pretreated with Mel significantly improved CCl4-induced liver fibrosis.


Introduction
The largest solid organ and digestive gland in the body is thought to be the liver, which plays a key role in purifying toxic chemicals, synthesizing new molecules, and storing and metabolizing nutrients. Although the liver has a strong regenerative capacity, subacute injury induced by internal and external factors can lead to liver fibrosis (Liu et al. 2018. Marslin et al. 2018. Carbon tetrachloride (CCl 4 ) is widely used in daily life. It is known to be used as a solvent for oils and fats, a refrigerant, a fumigant, a dry cleaner for fabrics, a metal cleaner, and an insecticide. The classic toxicity of carbon tetrachloride (CCl 4 ) is to induce liver lesions and liver fibrosis. For example, CCl 4 attack normal liver cells, resulting in liver cell damage, endothelial barrier damage, activation of inflammatory cells and hepatic stellate cells (HSCs), and damage to normal liver structure and function (Pedroza et al. 2019). Eventually, this leads to the occurrence of liver fibrosis. To date, some studies have indicated that abnormal activation and proliferation of HSCs are the key factors leading to liver fibrosis. On the one hand, upon activation of TGF-b1, HSCs are transformed into myofibroblast-like form and rapidly proliferate to over-synthesize ECM proteins such as collagen, proteoglycan, and fibronectin for the establishment of connective tissues, thereby promoting liver fibrosis (Cho et al. 2010). In addition, TGF-b1 inhibits the degradation of ECM by regulating matrix metalloproteinase (MMP), tissue inhibitors of metalloproteinases (TIMP), which inhibits MMP, and plasminogen activator inhibitor (PAI) (Dud as et al. 2001). MMP2 and MMP9 are closely related type IV collagenases that help shape the ECM (Chandra et al. 2021). Studies have shown that CCl 4 can activate TGF-b1/Smad2/3 signaling pathway, proinflammation, and oxidation, and activate MMP2 and MMP9 to stimulate ECM degradation (Chen et al. 2020). On the other hand, the intracellular hepatic sinus pressure is continuously increased by cell contraction, resulting in hyperplasia of collagen fibers. These two changes ultimately promote the formation of liver fibrosis (Sun and Kisseleva 2015). If left untreated, fibrous nodules can proliferate as the disease progresses, leading to disruption of normal liver structure and function. The disease can eventually develop into cirrhosis and even liver cancer. At present, liver transplantation is still an effective method for the treatment of liver fibrosis, but due to limited sources and severe immune rejection, it is difficult to obtain effective and extensive treatment (Fallowfield and Iredale 2004).
Stem cells are self-replicating, have the potential for multidirectional differentiation, and can constantly self-renew and transform into cells that form human tissues or organs under specific conditions. In recent years, stem cell transplantation has been regarded as a very effective alternative therapy for the treatment of liver disease due to its advantages and posttransplantation research (Zhang and Wang 2013). Stem cells (embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells) can differentiate into hepatocellular-like cells in vivo and in vitro. Among stem cell types, fat-derived stem cells (adipose-derived stem cells, ADSCs) are derived from adipose tissue and have the advantages of being abundant and easy to obtain, having strong proliferation ability, and good in vivo expansion (Strem et al. 2005, Zuk et al. 2001. In addition, ADSCs have immunomodulatory characteristics that allow them to migrate to the injured tissue area and secrete nutrients, such as growth factors and cytokines, which promote the repair and regeneration of damaged organs and tissues, including the liver (Eom et al. 2015). Previous studies have found that the abnormally elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum gradually decreased and became normal after transplantation of ADSCs into CCl 4 -induced liver fibrosis mice, indicating that ADSCs have an obvious protective effect on the damaged liver (Banas et al. 2009). Although ADSC transplantation has many positive effects in the treatment of liver fibrosis, there are still some deficiencies in ADSC transplantation. For example, the survival rate of transplanted ADSCs is relatively low and is greatly affected by the microenvironment in vivo; thus, a good microenvironment is needed to improve the therapeutic effect of ADSCs. Induced differentiation of hepatocyte-like cells in vitro still cannot be compared with mature liver cells. Therefore, it is of great significance to study how to improve the survival rate of ADSCs after transplantation and how to improve their paracrine effect.
Melatonin (Mel) is secreted by the pineal gland at night and the gastrointestinal tract during the day (Margheri et al. 2012). It is responsible for a variety of physiological functions, including antioxidant, antiapoptotic, and proapoptotic activities (Shibo et al. 2017, Mortezaee 2018, Mortezaee and Khanlarkhani 2018. Numerous existing studies have shown that Mel can promote the proliferation and differentiation of stem cells to some extent. For example, Kumar et al. found that Mel can promote the growth and proliferation of embryonic stem cells (ESCs) (Kumar et al. 2012). Fu et al. showed that Mel can promote the proliferation of neural stem cells (NSCs) under hypoxic conditions (Fu et al. 2011). Mel also plays a vital role in the differentiation, survival, and migration of NSCs (Jiaqi et al. 2016). In addition, BMMSCs pretreated with Mel achieved better therapeutic effects in myocardial infarction, cerebral ischemia, and renal ischemia models (Tang et al. 2014). Mel can promote the differentiation of dental pulp stem cells (DPSCs) into hepatocytes and help to reverse liver fibrosis (Cho et al. 2015). Here, we incubated ADSCs with Mel for 24 h to explore their therapeutic effect on CCl 4induced liver fibrosis.

Cell isolation and culture
Adipose tissue was isolated from the groin of 3-weekold mice and minced under sterile conditions. Next, the separated adipose tissues were digested with 0.1% collagenase at 37 C for 60 min, and the digestion was terminated with 10% FBS. After centrifugation, it was added to the medium and cultured in an incubator for 24 h, and then, the medium was replaced with fresh medium to remove unattached cells and impurities. ADSCs from mice were subcultured twice before being used in experiments (DMEM; FBS; Gibco; Thermo Fisher Scientific, Inc.).

Preconditioning of ADSCs with Mel
When ADSCs were cultured to the third generation to 90% fusion, DMEM/F12 culture medium containing 50 nmol/L Mel was replaced, and cells were collected for treatment after 24 h of culture (Gao et al. 2014, Radio et al. 2006).

Animals
Male Kunming mice (n ¼ 40) weighing 20 ± 2 g were purchased from the Animal Center of Medical College of Henan University of Science and Technology (Luoyang, Henan, China). All mice were placed in an air-conditioned room with a temperature of 24 C and a light cycle of 12 h and were provided with free-feeding standard laboratory water and food. All animal experiments were approved by the Animal Protection and Use Committee of Henan University of Science and Technology. Forty mice were randomly divided into a control group, model group, ADSC group, and ADSC þ Mel group, with 10 mice in each group. The model group, ADSC group, and ADSC þ Mel group mice were intraperitoneally injected with 8% CCl 4 solution at 1 ml/kg body weight (CCl 4 :oil ¼ 8:92) to induce liver fibrosis and the control group was injected with the same dose of PBS twice a week for 4 weeks. Starting from week 5, mice in the ADSC group and ADSC þ Mel group were injected with 1 ml PBS cell suspension containing 1 Â 10 6 ADSCs or ADSCs pretreated with 50 nM Mel twice a week for 2 consecutive weeks. The mice in the model group and the control group were injected with the same dose of PBS via the tail vein.

Biochemical assays
Blood was extracted from the heart immediately after the mice were sacrificed, and the serum was separated via centrifugation at 4 C and 3500 rpm for 10 min and stored at À20 C. The activities of ALT, AST, ALP, and ALB in the serum samples were measured using a commercial kit (Nanjing Jiancheng Biotechnology Co., LTD, Nanjing, China) according to the manufacturer's instructions.

Histopathological analysis
After the liver tissue was fixed with 4% polyformaldehyde for 48 h, the tissue was dehydrated, embedded in paraffin, and sectioned at 5 lm. The sections were stained with hematoxylin and eosin (H&E), and histopathological changes were observed under a microscope. Paraffin sections were also collected to perform immunohistochemistry and Masson's trichrome staining. Three representative sections were selected from each liver tissue and were analyzed in turn.

Quantitative real-time reverse transcription PCR (RT-qPCR)
Briefly, TRIzol (Cwbio Technologies, Beijing, China) reagent was used to extract total RNA from cryopreserved liver tissue when qRT-PCR analysis of liver tissue was performed. Next, cDNA reverse transcription was performed using a PrimeScript RT reagent kit (Takara, Dalian, China). A SYBR V R Premix Ex Taq TM kit (Takara, Dalian, China) was used for quantitative PCR assays. Target gene expression was quantified using the 2 2᭝᭝ Ct method and normalized to the expression of GAPDH. The primer sequences are summarized in Table 1.

Hydroxyproline assay
According to the instructions, liver hydroxyproline (Hyp) levels were measured using a specific assay kit (Solarbio, Beijing, China). Five replicates were used for each test.

Immunohistochemical staining and assessment
Before the immunohistochemical study, liver tissue sections were fixed with 4% paraformaldehyde for 48 h, followed by tissue dehydration, paraffin embedding, and (5 lm) sectioning. After repair with 0.1 mol/l antigenic repair solution, 3% H 2 O 2 was used for the inactivation of endogenous enzymes, followed by treatment with 5% BSA as a sealant. The sections were incubated with primary antibodies targeting TGF-b (1:400 dilution), MMP-9 (1:800 dilution), and MMP-2 (1:200 dilution) overnight at 4 C and then with goat anti-rabbit/mouse HRP-labelled secondary antibodies at 37 C for 30 min. After dehydration, the tissue was sealed with neutral gum and observed under a Nikon Eclipse E100 microscope. The immunohistochemical results were measured using Image J software to determine section optical density (IOD).

ELISA analysis
The isolated sera were detected according to the levels of TNF-a and IL-6, respectively, according to the ELISA kit (Sangon Biotech, Shanghai, China) operating procedure instructions. The gradient diluted standard antibody was added to the sample to be tested, incubated at 37 C for 60 min, and then washed 5 times. A and B chromogenic agents were added for 15 min of dark color development, and the reaction was terminated with a termination solution. Measured absorption value (OD450nm).

Statistical analysis
With SPSS 20.0 statistical software, the data were analyzed using an LSD (least significant difference) posthoc test and one-way ANOVA. All the data are presented as the means ± SD. p < 0.05 was considered statistically significant.

Serum biochemical analysis
Serum ALT, AST, ALP and ALB levels were detected to observe changes in liver function. As shown in Figure 1, it is obvious that the serum levels of AST, ALT, and ALP in the model group were significantly higher than those in the control group (p < 0.01), while the level of ALB showed the opposite trend (P < 0.01). After treatment with ADSCs, we found that the AST, ALT, and ALP activities in serum from the ADSC group and ADSC þ Mel group mice were significantly reduced compared with levels in the model group (p < 0.05), and ALB levels were significantly increased (p < 0.05). Notably, the changes in the Figure 1 The effect of Mel-incubated ADSCs on CCl 4 -induced serum biochemical indices. Note: Ã indicates significance, p < 0.05; ÃÃ indicates extreme significance, p < 0.01.
These results indicate that mel-pretreated ADSCs are more helpful to CCl 4 -induced liver injury.

Mel-incubated ADSCs improve CCl 4 -induced liver pathological changes
Liver tissue was stained with H&E for histopathological analysis. The hepatic sinusoids and hepatocytes of livers from the control mice were arranged radially around the central vein, with an intact structure and large and round nuclei ( Figure 2). Compared with the above findings, unclear outlines, disordered arrangement, severe cytoplasmic vacuolation, and fragmented nuclei were found in the model group ( Figure 2). The histological changes of the liver recovered significantly after treatment. We found that compared with the ADSCs group, the recovery of liver pathological changes was better in the Melincubated ADSCs group. The above results indicate that Mel-incubated ADSCs had a better effect on the pathological changes in mouse livers induced by CCl 4 .

Mel-incubated ADSCs improve CCl 4 -induced liver fibrosis
To explore the effect of Mel-incubated ADSCs on liver fibrosis, Masson staining and detection of Hyp content were used to reflect the collagen fiber content in livers from each group. Collagen fibers were dyed green. As shown in Figure 3, compared with the control group, the green area and Hyp content in the model group were significantly increased (p < 0.01), indicating that the liver had obvious fibrosis. After 2 weeks of treatment with ADSCs, the fibrotic area and Hyp content were significantly reduced (p < 0.01). It was noted that compared with the ADSC group, the Melincubated ADSCs had a better effect in reducing collagen fibers (P < 0.01). The above results indicate that Mel-incubated ADSCs had a better therapeutic effect on CCl 4 -induced liver fibrosis in mice.

Mel-incubated ADSCs improve CCl 4 -induced hepatocyte apoptosis
The Bcl-2 and Caspase families play an indispensable role in the execution and regulation of apoptosis. The expression levels of the proteases caspase-8, Bax, and caspase-3; the antiapoptotic gene Bcl-2; and the proapoptotic gene Bax in each group were detected via qPCR. As shown in Figure 4, compared with the control group, the expression levels of Bcl-2 were significantly reduced and the expression levels of Caspase-8, Bax, and Caspase-3 were significantly increased in the model group (p < 0.01). After treatment with ADSCs, the expression level of Bcl-2 was significantly increased (p < 0.01), and the expression levels of Caspase-8, Bax, and Caspase-3 were significantly decreased (p < 0.01).
Notably, the changes in the ADSC þ Mel group were more significant. The above results indicate that Melincubated ADSCs had a better therapeutic effect on CCl 4 -induced hepatocyte apoptosis.
3.5. Mel-incubated ADSCs regulated the expression of MMP-9, TGF-b and MMP-2 in liver injury The occurrence of liver fibrosis is mainly caused by excessive synthesis or reduced degradation of the

Mel-incubated ADSCs regulated the expression of TNF-a and IL-6 in liver injury
The expressions of inflammatory factors TNF-a and IL-6 in the liver were detected by ELISA, as shown in Figure 8. Compared with the control group, the model group showed a high level of inflammatory expression (TNF-a and IL-6 levels and mRNA expression levels were significantly increased (p < 0.01)). After treatment, the expression of inflammatory factors (TNF-a and IL-6) decreased significantly (p < 0.05), the ADSC group pretreated with Mel had the most significant decrease in inflammatory factors (p < 0.01).

Discussion
In recent years, CCl 4 -induced liver injury has been studied in many aspects, and liver fibrosis is the most widely studied in clinical practice. To date, the clinical treatment methods for liver disease are divided into drug therapy (Lambrecht et al. 2020) and liver transplantation. The unreasonable use of drugs will produce side effects; for example, 7%-29% of patients will develop toxemia due to reasonable and unreasonable use of statins. Excessive use of acetaminophen will cause a series of pathological phenomena (such as vacuolar degeneration, hepatic lobular necrosis, and hemorrhage), leading to liver failure, and adverse reactions to flavonoids and Chinese medicine saponins can also cause liver toxicity ( another study found that intrahepatic transplantation of stem cells can change immune regulation in the liver microenvironment (Regmi et al. 2019) and reduce the inflammatory damage related to liver disease (Chen et al. 2017); thus, in recent years, stem cell transplantation has become a research topic in the treatment of liver disease. For example, due to abnormal activation and proliferation of hematopoietic stem  cells leading to liver fibrosis, ADSCs can promote the expression of HGF in the liver, prevent the expression of a-SMA, inhibit the proliferation of HSCs, and promote the apoptosis of HSCs, thereby alleviating liver fibrosis (Tang et al. 2018). Yan et al. noted that stem cell transplantation can change the migration of MDSCs, CD4 þ T cells, and the expression of immune cytokines to prevent acute liver injury caused by ConA (Bi et al. 2019a). Recently, a study using ADSCs of mesenteric origin to treat CCl 4 -induced liver cirrhosis in rats for 5 weeks found that ADSCs could improve the histopathological changes in the liver and enable the rats to have normal clinical behavior (Nazhvani et al. 2020). Based on liver function, biochemical and pathological tests, we concluded that ADSC transplantation improves CCl 4 -induced hepatic fibrosis injury. Cells of adult organs can be replaced by differentiation or transdifferentiation of stem cells, especially after injury (Kopp et al. 2016). MSCs either lack the expression of class II major histocompatibility (MHC) antigens or many molecules (CD80, CD40, CD86N) required for immune recognition (Alfaifi et al. 2018); thus, low immunogenicity is used by many investigators in experiments. It is known that stem cells or specially differentiated cells transplanted at the site of liver injury can differentiate or transdifferentiate under the influence of cytokines released by various cells, and differentiated liver cells or other cells can provide support for the liver and promote liver tissue repair (Bandi et al. 2019, Ghosheh et al. 2017. Lay et al. found that human pluripotent stem cells (HPSCs) differentiated into hepatocyte progenitor cells on Day 6 and hepatocellular-like cells on Day 18, and they further showed that HPSC-derived hepatocellular-like cells transplanted into liver injury model mice migrated to the injury site, improving liver function and promoting the survival rate of mice (Ang et al. 2018, Loh et al. 2019. Gage et al. showed that human pluripotent stem cells (PSCs) could differentiate into venous angioblasts in vitro, which upregulated the  markers of hepatic sinusoid endothelial cells (LSECs) and produced mature LSECs after intrahepatic transplantation of LSECs in neonatal mice and adult mice (Gage et al. 2020). In addition, paracrine factors (such as cytokines, growth factors, and exosomes) of stem cells can promote indirect and remote liver tissue repair by indirectly mediating anti-apoptosis activity, regulating nutritional factors, promoting angiogenesis, and exerting anti-fibrosis, antioxidative, and immunosuppressive effects (Watanabe et al. 2019). For example, stem cell-derived extracellular vesicles accumulate mainly in the liver, kidney, spleen, and lung, and extracellular vesicles can reduce liver and kidney injury by reducing hepatic stellate cell activation and liver fibrosis or by treating mitochondrial injury through the mitochondrial transcription factor A (TFAM) pathway (Povero et al. 2019). Exosomes released into the extracellular environment by mesenchymal stem cells can be absorbed by target cells in the microenvironment or can be carried far away through biological fluids and inhibit hepatic stellate cell (HSC) activation through the Wnt/b-catenin pathway, thereby modulating oxidative markers (MDA, SOD, GSH-Px and CAT) and liver function markers (Hyp) and improving CCl 4 -induced liver fibrosis (Lou et al. 2017, Ping et al. 2018, Rong et al. 2019. At the same time, the study of lung tissue fibrosis by Wang et al. showed that MSC significantly down-regulated the levels of MDA and MPO in lung tissue, increased the levels of GSH and SOD, reduced the apoptotic cells in lung tissue through antioxidant, and reduced the expression levels of tissue inflammatory factors (IL- 1b, IL-8, TNF-a, IL-10) (Wang et al. 2021). Su et al. showed that milk fat globule-EGF factor 8 (MFGE8), an antifibrotic protein in the MSC-secreted group and a potent inhibitor of HSC activation, strongly inhibited TGF-b signaling and reduced extracellular matrix deposition and liver fibrosis in mice .
However, stem cell transplantation has some problems, such as cell viability, differentiation, and survival barriers at the damaged site after transplantation, which make the efficiency of transplantation less than ideal (Kim et al. 2015). Adrien et al. showed that the energy needed by stem cells is produced by glucose fermentation and that stem cells die within three days of implantation when the damaged tissue area becomes too hypoxic (0.1% O 2 ) to produce energy and the body's energy reserves are depleted (Moya et al. 2018). It has been reported that cell necrosis and apoptosis may occur after treatment with stem cells due to the loss of adhesion-related signals in transplanted cells (Don and Murry 2013). Other studies have shown that ROS normally controls the signal transduction mechanism of transplanted stem cells, but under pathological conditions, tissue damage produces a large amount of ROS, which harms the survival of stem cells (Kr€ ankel et al. 2011). A 2010 study showed that one hour after treating ischemic injury with stem cells, less than 1% of the cells were detected in the injured kidney, and the cells disappeared within a few days of injection (Burst et al. 2010). As research continues, it was reported in 2019 that within a week of transplantation, more than 80% to 90% of the transplanted stem cells die, leaving 9% to 19% of the cells in the animal (Zhao et al. 2019). Although transplant survival rates have improved, they are still insufficient to meet demand, and more research is needed. Therefore, overcoming these obstacles is the key to stem cell transplantation for the treatment of liver disease.
Mel has been used by many researchers to improve the efficiency of stem cell proliferation, differentiation, and transplantation in clinical experiments due to its anti-inflammatory, antiaging, antioxidative, and antiapoptotic activities and other properties. For example, some scholars have found that melatonin can promote the proliferation and osteogenic differentiation of stem cells through the PI3K/Akt and SIRT1/SOD2 signaling pathways in vitro (Liao et al. 2020, Liu et al. 2020, Yu et al. 2019, Zhang et al. 2020. Mel also has a protective effect on the aging of BMSCs induced by iron overload because it can promote the proliferation of BMSCs and reduce ROS storage and the expression of ERK/p53/p38 (Yang et al. 2017). In addition, studies have shown that Mel can reduce aging and improve the survival rate of ADSCs by inhibiting the NF-jB signaling pathway (Fang et al. 2018) and enhancing mitochondrial function (Lee et al. 2020b). In treating kidney disease, Saberi et al. transplanted Mel-pretreated BMSCs into damaged kidneys and found that they reduced renal fibrosis, improved the condition of the damaged renal tubule tissue, and significantly increased cell transplant survival (Saberi et al. 2019). Li et al. demonstrated that Mel-pretreated MSCs enhanced mitochondrial Mel and cell cyclin-related protein activity by increasing the expression of peroxidase-associated receptor PGC-1a, thereby significantly increasing the survival rate of MSCs, promoting the formation of new blood vessels, decreasing fibrosis, and improving the ischemic tissue in limbs (Lee et al. 2020a). In the treatment of CCl 4 -induced hepatic fibrosis, Mel-pretreated BMSCs decreased the expression of Bax and TGF-b1 and the serum ALT content but increased the expression of Bcl2 and MMPs compared with BMSCs alone . According to our previous study, 50 nM MT at different concentrations significantly promoted the proliferative activity of ADSCs, and thus, we used 50 nM MT-pretreated ADSCs to treat CCl 4 -induced liver fibrosis. The results showed that ADSCs pretreated with 50 nM MT significantly improved liver function, liver pathology, and apoptotic gene expression compared with ADSCs alone.
The synthesis and degradation of the extracellular matrix (ECM) are in dynamic equilibrium in the normal liver. When the liver is damaged, this equilibrium is disrupted, leading to activation of fibrosis-related matrix metalloproteinases (MMPs), which are responsible for the turnover of ECM during highly dynamic processes (Bi et al. 2019b). In addition, it has been reported that the acute phase of fibrosis progression leads to increased expression of matrix metalloproteinases (MMPs) and tissue inhibitors (TIMPs) in the liver (Arthur 2000). However, the expression of MMP-2 and MMP-9 is considered an early signal of hepatotoxicity and a hepatitis response (Hernandez-Gea and Friedman 2011). Studies have also shown that fibrosis leads to abnormal activation and proliferation of hematopoietic stem cells (HSCs), and decomposition of type IV collagen by activated MMP-2 is a necessary condition for HSCs to maintain a physiological static state (Ma et al. 2019). Activated HSCs are also considered to be the primary source of MMP-2 in the liver, which in turn promotes the activation of HSCs by degrading the normal subcutaneous matrix and disrupting the environment of HSCs and other cells (Ma et al. 2019). Therefore, the production of MMP-2 interacts with the activation of HSCs. Similar to MMP-2, activated MMP-9 also degrades type IV collagen, which in turn promotes the activation of HSCs and Kupffer cells, which secrete large amounts of TIMPs, type I collagen, and type III collagen (Wang et al. 2019, Xu et al. 2014. For example, Long et al. showed that CCl 4 -induced liver injury can significantly increase the expression of MMP-2 and MMP-9, while trans-2,3,5,4 0 -tetrahydroxystilbene 2-O-b-D-glucopyranoside (Trans-THSG) significantly reduced this change (Long et al. 2019). Artesunate has also been shown to improve bovine serum albumininduced liver fibrosis in rats by decreasing MMP2, a-SMA, and MMP-9 expression (Xu et al. 2014). During the development of liver fibrosis, some endogenous profibrogenic factors, such as IL-17, are released, which in turn stimulate Kupffer cells to produce other proinflammatory factors (TGF-b1 and TNF-a), all of which stimulate HSC activation to express collagen and enhance ECM deposition (Long et al. 2019). In addition, studies have shown that TGF-b is activated through a variety of mechanisms, including MMP2 and MMP-9 (Piek et al. 1999). Li et al. also verified that the Chinese medicine CGA formula can reduce the expression of TGF-b, MMP2, and MMP-9 in the liver tissue of rats with DMN-induced liver fibrosis (Li et al. 2016). Again, our study found that MMP2-, TGF-b-and MMP-9-positive cells were significantly expressed in the liver after administration of CCl 4 , and the number of these cells was significantly reduced by Mel-incubated ADSCs. At the same time, our results showed that Mel-pretreated ADSC also significantly reduced the expression levels of inflammatory factors in mice. It is suggested that reducing the expression of MMP2 and MMP-9 can improve the environment for the survival of HSCs and other cells, thereby reducing the activation of HSCs and improving pathological changes in liver tissue. However, the role of MMP2 and MMP-9 remains controversial. Daniel et al. showed that MMP2 and MMP-9 were significantly expressed in rat liver fibrosis induced by diethylnitrosamine, while treatment with S-nitroso-N-acetylcysteine (SNAC) significantly reduced this change (Mazo et al. 2013). These conflicting results may be due to the use of different types of animal models and to the very complex mechanisms leading to liver fibrosis. Therefore, exploration of the mechanisms underlying liver fibrosis may require further research.

Conclusion
In conclusion, mel pretreatment of ADSCs significantly reduced the expression levels of inflammation, apoptotic genes and liver fibrosis related indicators (MMP-2, MMP-9 and TGF-b) after treatment, reduced the area of liver fibrosis induced by ccl4, and effectively improved the degree of liver fibrosis in mice. This provides a reference for the clinical application of mesenchymal stem cells in the treatment of the liver injury.