Cell culture
hFSSCsand 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. In brief, hFSSCs, hUCMSCs and HSCs were cultured in DMEM (Gibco, Grand island, U.S.) supplemented with 500 U/ml penicillin and 500 μg/ml streptomycin (Invitrogen, Shanghai, China), and 10% FBS (Gibco, Grand island, U.S.) at 37°C, with saturated humidity and 5% CO2. hFSSCs and hUCMSCs at the P5 were used for this study, and hFSSCsand hUCMSCs) secretome was collected as reported in our previous study[13].Briefly, cells were cultured and reached 70~80% confluence, placed in serum-free medium (SFM; Invitrogen, Shanghai, China), and incubated in 5% CO2 in a humidified condition. After cultured 24h, the conditioned medium (CM) was collected and centrifuged to purify for 10 min at 4 °C, 4000g. Next, 10 ml conditioned medium was re-centrifuged with Amicon Ultra Centrifugal Filters (Millipore Corp, Billerica, MA, USA) at 4 °C, 4000g, 2 h. At last, 300~500 μl supernatant solution was collected as cell-free secretome each time. The protein content was measured using the BCSA kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instruction.
CCl4-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 Traditional Chinese Medicine (Approval NO. XW201903167). Detailed procedures for CCl4-induced have been described in our published studies[6]. Briefly, rats were administered with an intraperitoneal injection of 30% CCl4, 3ml/kg body weight twice weekly in olive oil. After eight weeks, CCl4 treated rats were randomly assigned into three groups (n=10 rats, tail vein injection/weekly): PBS group (1ml); hUCMSCsecretome group (250μg, 1ml); hFSSCsecretome 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 and Sirius redaccording 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% H2O2 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), α-SMA (ab5694, 1:500 dilution, Abcam, Cambridge, UK), and HNF-4α(ab219610, 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.
Biochemical analysis
The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT) were assessed using the Automated Biochemical Analyzer (AU-680, Beckman, California, U.S.) according to the procedure. Liver homogenate (10%, w/v) was prepared by homogenizing the right lobe of liver on ice in 150 mMTris-HCl buffered saline (pH 7.2) using a polytron homogenizer (PT3100D; Kinematical, Lucerne, Switzerland). The levels of Malondialdehyde (MDA) and Hydroxyproline (Hyp) in liver tissue were measured using kits (NanJingJianCheng Bio., Nanjing, China) according to the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
HSCs were co-cultured with either PBS, hUCMSCsecretome, orhFFSCsecretome(5ng/ml) for 48h before samples were collected for mRNAextraction.Total RNA was isolated from HSCs 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 levels of EMT related genes (E-cadherin, Snail1, Vimentin, FSP1 and α-SMA) ,TGF-β/Smad signaling pathwayrelated genes (TGF-β1, Smad2, Smad3, Smad7 and Collagen I),and the internal standard GAPDH mRNA were measured by qRT-PCR.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.
Immunofluorescence (IF) staining
When HSCs reached 60~70% confluence on 24-well plates, they were cultured with with either PBS, hUCMSCsecretome, orhFFSCsecretome (5ng/ml) for 48h. Next, HSCs were incubated with 4% paraformaldehyde at room temperature for 10 minutes, and then incubated with 1% bovine serum albumin (BSA, Biosharp, Wuhan, China) for 30 minutes. Cells were incubated with a primary antibody against α-SMA (ab5694, 1:100 dilution, Abcam, Cambridge, UK) for 1h, followed by incubation with a secondary antibody (goat anti-rabbit IgG, ab15007, 1:500 dilution, Abcam, Cambridge, UK) for 30 minutes at room temperature. Rhodamine phalloidin (Thermal Scientific, Waltham,U.S.) was stained for cytoskeleton. The nuclei were labeled with DAPI (Thermal Scientific, Waltham,U.S.). Fluorescent images were captured using an EVOS Cell Imaging System (Thermo Scientific, Waltham, U.S.).
Western blotting
HSCs were co-cultured with either PBS, hUCMSCsecretome, orhFFSCsecretome (5ng/ml) for 48h before samples were collected for protein extraction. Protein samples were mixed with SDS sample buffer and heated to 95 °C for 10 minutes, followed by separation on SDS-polyacrylamide gels. Resolved proteins were electro-blotted onto nitrocellulose membrane and probed with antibodies againstTGF-β1(ab92486), Smad2(ab40855), Smad3(ab40854), Smad7(ab216428), Collagen I (ab90395) andβ-actin(ab5694), (1:1000 dilution, Abcam, Cambridge, UK)overnight at 4℃ (1:1000 dilution, Abcam, Cambridge,UK). Nitrocellulose membranes were then incubated with a secondary antibody, HRP-conjugated goat anti-rabbit IgG (ab15007), at room temperature for 2h, and visualized by chemiluminescent detection according to the manufacturer’s instructions (Immobilon western chemiluminescent HRP substrate, Millipore, Massachusetts, U.S.).
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.for at least three independent experiments, and p < 0.05 was considered to be statistically significant.
Results
hFSSCsecretome reduced CCl4-induced liver fibrosis in rats.
To explore the effect of hFSSCsecretome on liver fibrosis, we using CCl4 -induced a liver fibrosis model in rats (Fig. 1a). Compared to the PBS group, gross morphology changes obviously in hFSSCsecretome 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 Massonand Sirius red staining indicated that the collagen areapercentage in hFSSC group (9.2%) was significantly reduced, compared to the other two control groups (24.3% in PBS group and 14.9% in hUCMSCsecretome group, Fig. 1band 1c, p<0.05).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 hFSSCsecretome group was significantly lower than the other two control groups (Fig.1dand 1ep<0.01). These findings suggest that hFSSCsecretome effectively reduced CCl4-induced liver fibrosis in rats.
hFSSCsecretome reduced liver fibrosis through suppressing the EMT
To further verify the roles of hFSSCsecretom 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 hFSSCsecretome group reduced fluorescence intensity observably, compared to the other two control groups(Fig.2a).In future study, we explore the effect of hFSSCsecretome on EMT, and qRT-PCR analysis was used to examine the expression ofEMT-related indicators(E-cadherin, Snail1, Vimentin, FSP1 and α-SMA in HSCs).Interestingly, our results found that hFSSCsecretome treatmentincreased the epithelialmarker of E-cadherinexpression, while decreased the transcription factors of Snail and mesenchymal markers (Vimentin, FSP1 and α-SMA) expression, compared to the PBS group (Fig. 2b, p<0.05).Meanwhile, we also examined hFSSCsecretomeincreasedE-cadherin, and decreasedFSP1 and α-SMA expression compared to the hUCMSCsecretome group (Fig. 2b, p<0.05). These results suggest that hFSSCsecretome reduced liver fibrosis through suppressing the EMT.
hFSSCsecretome improved liver functionality and promoted liver regeneration
To explore the effect of hFSSCsecretome on liver functionality, we performed the biochemical analyses. In comparison to the PBS group, hFSSCsecretome 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 inhFSSCsecretome group was higher than that inPBS group (Fig. 3f, p<0.05). In addition, hFSSCsecretome group significantly reduced the serum levels of TBIL and γ-GT compared to hUCMSCsecretome group (Fig. 3c and 3d, p<0.05). These results suggest that hFSSCsecretome effectively improved liver functionality.
Next, we performed IHC to assesse the effects of the hFSSCsecretome 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 areainhFSSCsecretome group (0.82%) was significantly decreased compared to the PBS group (5.51%, Fig. 4a and 4b, p<0.001). PCNAand HNF-4αis two crucial indicator of hepatocyte proliferation.IHC results showed that the percentage of PCNA positive areainhFSSCsecretome group (4.13 %) was significantly increased compared to the PBS group (7.48%, Fig. 4a and 4c, p<0.01).Consist with the above results, the percentage of HNF-4αpositive areawas significantly increased inhFSSCsecretome group (12.1%), compared to the PBS group (4.5%,p<0.01) as well as hUC-MSCs group (8.2%, Fig. 4a and 4d, p<0.05).The histological results indicated that hFSSCsecretome effectively delayed the progression of liver fibrosis, and promoted the liver regeneration.
hFSSCsecretome regulate the TGF-β/Smad signal pathway
To investigate the underlying mechanism of the effect of hFSSCsecretome 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 hFSSCsecretome group, compared that of PBS group (Fig. 5a and 5b, p<0.001). However, we detected the Smad7 was significantly increased in hFSSCsecretome 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 hFSSCsecretome effectively reduced liver fibrosis viaregulating the TGF-β/Smad signal pathway (Fig5).