Human fetal skin-derived stem cell secretome reduce liver fibrosis by regulating TGF-β/Smad signal pathway CURRENT STATUS:

Background: Liver fibrosis resulting from chronic liver injury is one of the major causes of mortality worldwide. Stem cells-secreted secretome has been evaluated for overcoming the limitations of cell-based therapy in hepatic disease, while maintaining its advantages. Methods: In this study, we investigated the effect of human fetal skin-derived stem cells (hFFSCs) secretome in the treatment of liver fibrosis. To determine the therapeutic potential of the hFFSCs secretome in liver fibrosis, we established the CCl4-induced rat liver fibrosis model, and administered hFFSCs secretome in vivo. Moreover, we investigated the anti-fibrotic mechanism of hFFSCs secretome in hepatic stellate cells (HSCs). Results: Our results showed that hFFSCs secretom effectively reduced collagen content in liver, improved the liver function and promoted liver regeneration. In addition, we found that hFSSC secretom inhibited the TGF-β1, Smad2, Smad3, and Collagen I expression, however, increased Smad7 expression. Conclusions: In conclusions, our results suggest that hFFSCs secretome treatment could reduce CCl4-induced liver fibrosis via regulating the TGF-β/Smad signal pathway.


Introduction
Liver fibrosis is a wound healing response generated against chronic or iterative liver injury [1]. Recent evidence suggests that stem cell-based liver fibrosis treatment can be mediated through paracrine effects [2,3]. The exclusive use of stem cell-secreted secretome, has been evaluated for overcoming the limitations of cell-based therapy, while maintaining its advantages to their parent cells [4]. It included that extracellular vesicles, and other soluble proteins or biologically active molecules. In addition, previous studies have indicated that human bone marrow mesenchymal stem cells-derived exosomes and other stem cells-derived secretome can recued liver fibrosis [5,6].
Previous studies have indicated that the features of fetal tissue cells facilitate engraftment in vivo and may provide preferred effects against diseases difficult to treat [7]. Since 1928, hundreds of clinical trials using various types of fetal transplants have been performed worldwide [8][9][10]. Moreover, recently studies have been demonstrated that human fetal stem cells have a great growth promoting 3 potential, which benefited to the tissue regeneration and cell therapy [11,12]. In comparison to other mesenchymal stem cell (MSCs), fetal stem cells (FSCs) are easier to culture and more readily proliferate, and less likely to be rejected by transplant recipients, as these cells are less antigenic [8].
Although there remain still ethical and social issues with respect to the clinical use of fetal tissue, fetal stem cell secretome transplantation may overcome these problems and have more perspectives on hepatic disease treatment.
In our previous study, we have successfully isolated and identified hFSSCs [13]. Interestingly, we found hFSSC secretome has great ability to control and balance the collagen formation in skin tissue [13]. Therefore, we hypothesis that hFSSC have a potential to reduce the collagen formation in liver fibrosis, whereas liver fibrosis is caused by over-abundance of collagen. We further analyzed the effects of hFSSC secretome on live fibrosis in vitro, and the investigated the mechanism of hFSSC secretome and TGF-β/Smad pathway involvement on anti-fibrosis. Our preliminary results provide the first evidence that hFFSCs secretome effectively reduce liver fibrosis through the TGF-β/Smad pathway. We believe that the hFFSCs secretome as an acellular regenerative therapy and approaches can provide great potential for the treatment of liver fibrosis.

Cell culture
hFSSCs and human umbilical cord mesenchymal stem cell (hUCMSCs) were provided and extracted by our previous study [13]. HSCs were purchased from the Chinese Academy of Medical Sciences, China.

CCl 4 -induced liver fibrosis in rats
Liver fibrosis was induced in Sprague Dawley (SD) rats (8-week old, female, 200g). All protocols and procedures were approved by the Animal Experiment Ethic Committee of Changchun University of 4 Traditional Chinese Medicine (Approval NO. XW201903167). Detailed procedures for CCl 4 -induced have been described in our published studies [6]. Briefly, rats were administered with an intraperitoneal injection of 30% CCl 4 , 3ml/kg body weight twice weekly in olive oil. After eight weeks, CCl 4 treated rats were randomly assigned into three groups (n=10 rats, tail vein injection/weekly): PBS group (1ml); hUCMSC secretome group (250μg, 1ml); hFSSC secretome group (250μg, 1ml). After 4 weeks, liver tissue and serum were collected. Livers were divided into two parts of preservation in 10% formalin and freezing at -80 °C.

Histopathological analysis
Liver tissues were processed for paraffin embedding by slicing into 4μm sections. Liver sections were stained with hematoxylin & eosin (H&E) and Masson according to standard protocols. We selected the liver section fields randomly to analyze the liver fibrosis. The percentage of collagen stained area was calculated via Image-Pro Plus. Immunohistochemistry (IHC) was measured with the Kit (Maixin KIT-9710, Fuzhou, China) in accordance with the manufacturer's instructions. In brief, the liver sections were deparaffinized, rehydrated, and incubated in a 99 °C water bath for 15 minutes. Then, the slide was incubated with 3% H 2 O 2 for 15 minutes, and blocked with 10% normal goat serum for 1 h at 37°C . Following with the incubation of primary antibody against PCNA (ab15497, 1:500 dilution, Abcam, Cambridge, UK) and α-SMA (ab5694, 1:500 dilution, Abcam, Cambridge, UK) overnight at 4 °C. Next, slides were incubated with biotinylated goat-anti-rabbit IgG antibody. Add diaminobenzidine solution for 15 minutes at 37 °C, then incubated with avidin peroxidase reagent, and hematoxylin for counterstaining. Lastly, slides were photographed using an optical microscope (Olympus, Tokyo Metropolitan, Japan). We used 10 random fields per section and 10 sections in total (n=10 rats) for quantification of IHC results. The IHC results were calculated via Image-Pro Plus.

Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from liver tissue using Trizol reagent (Invitrgen, Shanghai, China) according to the manufacturer's protocol. Then, 1μg total RNA was reverse-transcribed to give cDNA, which was used as the template, and combined with standard SYBR premix Ex Taq (Invitrogen, Shanghai, China) on the Real-Time PCR Detection System (Roche, Basel, Switzerland), and experiments were conducted in triplicate. The primers are listed in Table S1, and GAPDH served as the internal control.
All reactions were performed in triplicate and the data were analyzed using the 2 -ΔΔCt method.

Statistical analysis
Statistical analysis was performed using GraphPad Prism Version 6. One-way ANOVA with Dunnett's multiple comparisons test was used to test for statistically significant differences. All quantitative data are expressed as mean ± SD. p < 0.05 was considered to be statistically significant.

hFSSC secretome reduced CCl 4 -induced liver fibrosis in rats.
To explore the effect of hFSSC secretome on liver fibrosis, we using CCl 4 -induced a liver fibrosis model in rats (Fig. 1a). Compared to the PBS group, gross morphology changes obviously in hFSSC secretome group, including less fibrous nodular and more ruddy on the surface, more uniform surface and soft texture (Fig. 1b). After 4 weeks treatment, histopathologic analysis using Masson staining indicated that the collagen area percentage in hFSSC group (9.2%) was significantly reduced, compared to the other two control groups (24.3% in PBS group and 14.9% in hUCMSC secretome group, Fig. 1b and 1c, p <0.05).
To further verify the roles of hFSSC secretom in the pathogenesis of liver fibrosis, we performed immunofluorescence staining of TGF-β in HSCs. As TGF-β1 is considered as a crucial mediator in tissue fibrosis, and HSCs are one of the major effector cells in liver fibrosis. We found that hFSSC secretome group reduced fluorescence intensity observably, compared to the other two control groups. Furthermore, we detected the MDA (a marker for oxidative stress and liver cell injury) and Hyp (a main component in collagen tissue) content in the liver tissue. We found that the level of MDA and Hyp in hFSSC secretome group was significantly lower than the other two control groups (Fig. 2b   7 and 2c p<0.01). These findings suggest that hFSSC secretome effectively reduced CCl4-induced liver fibrosis in rats.

hFSSC secretome improved liver functionality and promoted liver regeneration
To explore the effect of hFSSC secretome on liver functionality, we performed the biochemical analyses. In comparison to the PBS group, hFSSC secretome group significantly reduced serum levels of ALT, AST, TBIL, γ-GT and ALP (Fig. 3a-3e, p <0.05). However, the serum level of TP in hFSSC secretome group was higher than that in PBS group (Fig. 3f, p<0.05). In addition, hFSSC secretome group significantly reduced the serum levels of TBIL and γ-GT compared to hUCMSC secretome group ( Fig. 3c and 3d, p<0.05). These results suggest that hFSSC secretome effectively improved liver functionality.
Next, we performed IHC to assesse the effects of the hFSSC secretome on the liver. α-SMA is an important indicator of the occurrence and development of hepatic fibrosis. IHC results showed that the percentage of α-SMA positive area in hFSSC secretome group (0.82%) was significantly decreased compared to the PBS group (5.51%, Fig. 4a and 4b, p<0.001). PCNA is an crucial indicator of hepatocyte proliferation. IHC results showed that the percentage of PCNA positive area in hFSSC secretome group (4.13 %) was significantly increased compared to the PBS group (7.48%, Fig. 4a and   4c, p<0.01). The histological results indicated that hFSSC secretome effectively delayed the progression of liver fibrosis, and promoted the liver regeneration.

hFSSC secretome regulate the TGF-β/Smad signal pathway
To investigate the underlying mechanism of the effect of hFSSC secretome on liver fibrosis, we performed the Western blot and RT-qPCR to analysis the expression of TGF-β1, Smad2, Smad3, Smad7 and Collagen I in HSCs, as it is one of the major effector cells in liver fibrosis. We found that TGF-β1, Smad2, Smad3, and Collagen I expression was significantly decreased in hFSSC secretome group, compared that of PBS group (Fig. 5a and 5b, p<0.001). However, we detected the Smad7 was significantly increased in hFSSC secretome group, compared that of the other two control groups ( Fig.5a and 5b, p<0.01). Smad7 serves as a negative feedback regulator of TGF-β1/Smad pathway, thereby protects against TGF-β1-mediated fibrosis (Fig. 6). These results suggest that hFSSC 8 secretome effectively reduced liver fibrosis via regulating the TGF-β/Smad signal pathway (Fig5).

Discussion
In this study, our results demonstrated that hFSSC secretom reduce liver fibrosis both in rats.
Moreover, our research illustrated that hFSSC secretom reduce liver fibrosis through regulating the TGF-β/Smad signal pathway in HSCs.
Recent evidence indicates that the mesenchymal stem cell secretome as an acellular regenerative therapy for liver disease [5,14]. The use of MSC secretome has been shown to have anti-fibrotic effects [14]. Moreover, IV injection of hucMSC-EVs decreased liver fibrosis, reduced apoptosis and mitigated liver damage induced by CCl 4 in mice [15,16]. In our study, we found that hFSSC secretom effectively reduced CCl 4 -induced liver fibrosis, and improved liver functionality in rats (Fig. 1).
Moreover, hFFSCs secretomes treatment is more effective than of hUCMSCs secretomes in resolving fibrosis, such as some indicators included collagen area, MDA, Hyp, TBIL, and γ-GT. The reasons that differences found between hUCMSCs and hFFSCs secretomes may be reported that hFFSCs secretomes contains more biological activity factors than hUCMSCs secretomes [13]. Our studies indicated that hFSSC secretom is an attractive emerging option for therapeutic applications as a therapeutic strategy for liver fibrosis.
Previous study that mesenchymal stem cell secretome inhibit HSC activation and promoted liver regeneration [17][18][19]. HSCs stimulate the production of a large amount of collagen fibers to form liver fibrosis. Moreover, positive expression of α-SMA can serve as a marker for HSCs activation [20]. Our results demonstrate that hFSSC secretom decreased α-SMA expression. PCNA is an crucial indicator of hepatocyte proliferation [15]. Our study demonstrate that the percentage of PCNA positive area was significantly increased after hFSSC secretom treatment. These results confirmed that hFSSC secretom promotes liver regeneration while reducing fibrosis.Despite our results confirming that hFSSC secretome had significantly reduced liver fibrosis and caused no detectable immunological responses, their exact mechanisms of action need further exploration.
TGF-β1/Smad pathway is an important pathogenic mechanism in tissue fibrosis [21,22]. Studies have shown that TGF-β1 is considered as a crucial mediator in tissue fibrosis and causes tissue scarring 9 largely by activating its downstream small mother against decapentaplegic (Smad) signaling [23].
In conclusion, we successfully investigated the role of hFSSC secretome on cutaneous liver fibrosis.
Our results demonstrated that hFSSC secretome can exert promoting effect of liver fibrosis via regulating the TGF-β/Smad signal pathway.

Consent for publication
Not applicable.

Availability of supporting data
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Competing interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
This work was supported by the National Natural Science Foundation of China (No.8762069).   hFSSC secretome reduced liver fibrosis by regulating the TGF-β/Smad signaling pathway.

Authors' Contributions
hFSSC secretom inhibited TGF-β1expression, and actived Smad7 expression which are activated by the binding of the TGF-β super family to the cell surface receptors. TGF-β1mediated tissue fibrosis via Smad2/Smad3 which the two major downstream regulator, while Smad7 serves as a negative feedback regulator of TGF-β1/Smad pathway, thereby protects against TGF-β1-mediated fibrosis. Furthermore, hFSSC secretom decreased the collagen I expression, thereby reduce liver fibrosis.

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