Sal was procured from Shanghai Tauto Biotechnology (Shanghai, China), the alanine transaminase (ALT), aspartate aminotransferase (AST), and hydroxyproline (Hyp) detection kits were acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). FN, type I collagen (Col I), Bax and Bcl-2 antibodies were obtained from Abcam (Cambridge, United Kingdom). JNK, p-JNK, PARP, and cleaved caspase 3 antibodies were purchased from Cell Signaling Technology (Massachusetts, United States). SphK1, SphK2, S1PR1, S1PR2, S1PR3, CD9, and tumor susceptibility gene 101 (TSG101) antibodies were procured from ImmunoWay Biotechnology Company (Suzhou, China). Antibody against GAPDH was provided by Proteintech (Wuhan, China). TdT-mediated dUTP nick end labeling (TUNEL) Apoptosis Assay Kit, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DPAI), and Cy3-labeled goat anti-rabbit IgG were all bought from Beyotime Institute of Biotechnology (Shanghai, China). The S1P Assay Kit (S1P-ELISA) was delivered by Echelon Biosciences (Salt Lake City, United States).

Male C57BL/6J, SPF grade mice weighing 20 ± 2 g, were supplied by Shanghai Sippr-BK laboratory animal Co. Ltd. (Shanghai, China). They were placed in the experimental animal center of Shanghai University of Traditional Chinese Medicine, China. The Committee on the Care and Use of Live Animals for Teaching and Research of the Shanghai University of Traditional Chinese Medicine sanctioned the animal experiments.

Following a week-long adaptive feeding, 45 male mice were divided into the control group included 15 mice, while the modeling group had 30. The modeling group animals were intraperitoneally injected with 15% CCl₄ (diluted with olive oil) at 2 ml/kg thrice a week, whereas those in the control group were administered an equal dosage of olive oil intraperitoneally. After a week of modeling, the modeling group was segregated into Model and Sal groups. The Model and Sal groups were continued to model with CCl₄ for 5 weeks, while the Control group was injected intraperitoneally with equal dose of olive oil. The Sal group animals were administered Sal (20 mg/kg) through gavage for 5 weeks. Simultaneously, those in the Control and Model groups were both treated with equal doses of ddH₂O. At the end of five weeks, the mice were sacrificed on anesthetizing with pentobarbital sodium, and the serum and liver tissue samples were preserved for subsequent experiments.

The instructions provided along with the kit were followed to detect ALT and AST levels in the mice serum.

Fixation of the liver tissues was carried out in 10% neutral formalin for 48 h. They were then dehydrated stepwise with ethanol, followed by xylene permeation, and finally paraffin-embedded. Sections of 4 μm thickness were prepared for HE and Sirius red staining.

Following the kit directions, the Hyp contents in liver tissues were determined.

Healthy volunteers and cirrhosis patients were selected for collecting human liver tissue and serum samples from the Department of Hepatobiliary Surgery, Renji Hospital, Affiliated to Shanghai Jiao Tong University. The Ethics Committee of Shuguang Hospital, Affiliated to Shanghai University of Traditional Chinese Medicine, sanctioned this study.

Wild and Model groups of five male mice each were formed. The Model group mice were injected with CCl4 intraperitoneally to create a liver fibrosis model. They were then used to isolate primary LSECs. Using density gradient centrifugation in combination with CD146 magnetic bead (Miltenyi Biotec, Germany) separation, the primary LSEC of mice was isolated as the earlier reports (Liu et al., 2011; Meyer et al., 2016). Mouse liver tissue was perfused in situ and digested using an enzyme solution of collagenase D and DNase (Roche, Germany). Density gradient centrifugation was performed using OptiPrep solution (Axis-Shield, Norway) and incubation with CD146 magnetic bead for 30 min at 4°C. Thus isolated LSECs were cultured in Col I-coated dishes along with ECM medium, comprising 5% FBS, 2% endothelial cell growth supplement, and maintained with 5% CO₂ at 37°C in a humidified incubator.

Propagation of human immortalized HSCs (LX-2) and mouse immortalized HSCs (JS 1) was carried out in DMEM, supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. The SK-HEP-1 cells, obtained from the Chinese Academy of Sciences, were grown in MEM, containing 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. All cell lines were maintained with 5% CO₂ in a humidified incubator at 37°C.

The lentiviral vectors containing human SphK1 (LV-SphK1) or empty plasmid (LV-Cr) were procured from HANBIO Biotech (Shanghai, China). A 24-well plate was used to culture SK-HEP-1 to a confluence of 30–50%. They were then cultured in a fresh medium containing lentiviral vectors (MOI = 30) and polybrene (5 μg/ml) for 24 h, and then in a fresh medium for further culturing, followed by incubation for 48 h. The transfection efficiency of cells was studied using an inverted microscope and were incubated with a medium containing puromycin (1 μg/ml) to eliminate the untransfected cells. The transfected cells were cultured further, and the culture medium was used to isolate exosomes.

SK-HEP-1 cells were cultured in 150 mm dishes to a confluence of 80–90%. They were then cultured in MEM with 10% exosome-free serum for 24 h. The supernatant was ultracentrifuged to obtain the exosomes. The supernatant was first centrifuged at 4°C, 300 ×g, for 10 min to eliminate cells. The supernatant was centrifuged again at 4°C, 2,000 ×g, for 10 min to eradicate dead cells. The supernatant was centrifuged at 4°C, 10,000 ×g, for another 30 min to eliminate cell debris. Eventually, the supernatant was ultracentrifuged at 4°C, 100,000 ×g for 70 min, and the precipitate collected was the exosomes. The thus isolated exosomes were resuspended in PBS or DMEM medium for subsequent in vitro studies.

A transmission electron microscope was utilized to study the morphology of isolated exosomes (van Niel et al., 2018). This was followed by detecting typical exosomal marker proteins, CD9 and TSG101, by Western blot (WB) analysis.

LX-2 and JS 1 were cultured in 60 mm dishes to a confluence of 80–90%. LX-2 cells were allocated to Cr-exo and Cirrhosis-exo groups and incubated with serum exosomes (30 μg/ml) from healthy volunteers and cirrhosis patients, respectively, to study the migration of LX-2 cells. Also, JS 1 cells were distributed into Cr-exo, M-exo, and Sal-exo groups and incubated with serum exosomes (30 μg/ml) from corresponding mice to detect JS 1 cells’ migration. The LX-2 cells were then divided into Cr-exo, SphK1⁺-exo, Cr-exo + Sal, and SphK1⁺-exo + Sal groups and incubated with 30 μg/ml exosomes of transfected SK-HEP-1 cell culture media and/or Sal (10⁻⁵ M) to study the LX-2 cells migration and activation.

LX-2 or JS 1 cells were cultured in Col I-coated migration cups to a confluence of 80–90%. Then, exosomes and/or Sal were added to the lower chamber, and with the same medium being added to the upper chamber as well. Incubation was carried out for 24 h, and the migration cups were then taken out and air-dried. Fixation of the cells on the lower surface of the migration cup was done using 4% paraformaldehyde and staining with crystal violet. The total number of cells that migrated to the bottom was recorded with an inverted microscope (Olympus, Tokyo, Japan) and evaluated using ImageJ software (NIH, United States).

The adhered LSECs were incubated in ECM medium comprising DiI-Ac-LDL (Yiyuan Biotech, Guangzhou, China) for 4 h in the dark. The cells were then rinsed thrice with PBS, fixed with 4% paraformaldehyde for 15 min, and rinsed again with PBS. The endocytosis of DiI-Ac-LDL by LSECs was studied using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

According to the manufacturer's instructions, sections of frozen liver tissue of 8 μm thickness were prepared and incubated with TUNEL.

The LSECs were collected following 3 days of culturing, rinsed with PBS, and fixed using 4% paraformaldehyde for 30 min at room temperature. The frozen sections of mice liver tissue were fixed with precooled acetone for 15 min. Once fixed, the cells and frozen sections were rinsed again with PBS and incubated using 0.1% X-Triton-100 for 15 min at room temperature to disrupt the membrane. They were permeabilized and blocked using 5% BSA at room temperature for 1 h and then incubated with respective primary antibodies at 4°C overnight. The following day, they were incubated with anti-rabbit IgG (Cy3-labeled) in the dark at room temperature for 1 h and rinsed again with PBS. The cells were then stained using DAPI and rinsed using PBS. The sample was sealed, and their fluorescence was studied with the help of an inverted fluorescence microscope.

RIPA lysate comprising complete Mini, PMSF, and phosphatase inhibitors was utilized to isolate liver tissues and cells' total protein. Protein quantification was performed with the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). An equivalent amount (30 μg) of proteins was electrophoresed by SDS-PAGE and transferred to PVDF membranes (Millipore, United States). The membranes were then incubated with blocking buffer (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 1 h and incubated with respective primary antibodies overnight at 4°C. The membranes were rinsed using PBS-T for 20 min and treated with anti-rabbit IgG or anti-mouse IgG for 1 h. The membranes were rinsed with PBS-T again, and the images were recorded by means of Odyssey imaging system (LI-COR, United Kingdom) and analyzed with the ImageJ software. A part of the membranes was also stained with ponceau after incubating with antibodies.

TRIzol total RNA preparation kit (Shanghai Sangon Biotech, Shanghai, China) was utilized to isolate the total RNA of cells and synthesized to cDNA with a ReverAid First Strand cDNA Synthesis Kit (Thermo, United States) following the manufacturer’s procedures. The cDNA was mixed with primers and SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) in a certain ratio; the relative expressions of mRNA were measured utilizing the ViiA 7 Real-Time PCR System (ABI, Carlsbad, CA, United States). Primer sequences used for detection of Sphk1, SphK2, S1PR1,2,3 were shown in Table 1.

The outcomes have been recorded in terms of mean ± standard error of the mean. The differences among groups were evaluated via t-test or One-way ANOVA using SPSS 21.0 software. Values of p < 0.05 were considered statistically significant.

Serum ATL and AST were detected to be considerably higher in the Model group than those in the Control group (p < 0.01) (Figure 1A), indicating obvious liver function damage due to CCl₄. Nevertheless, these changes were observed to be reversed by Sal (p < 0.05 or p < 0.01) (Figure 1A). HE staining revealed that CCl₄ resulted in collagen deposition, structural destruction, and inflammatory cell infiltration in liver lobules and portal areas. Sal treatment was found to remarkably alleviate these histopathological lesions (Figure 1B).

Besides, Sirius red staining revealed excessive collagen deposition in the Model group than that in the control group, and Sal was noted to lower the collagen deposition (Figures 1C,D) substantially. Then, the effect of Sal on liver fibrosis was further examined by identifying the Hyp content in liver tissue and the characteristic indicators of liver fibrosis, including FN, Col I, and α-SMA. The Hyp content in liver tissues of the Model group was found to be remarkably higher as compared with that of the control group. Sal treatment reduced the Hyp level (p < 0.01) (Figure 1E). Moreover, higher quantities of FN, Col I, and α-SMA proteins were expressed in the Model group, which were considerably subdued by Sal administration (p < 0.05 or p < 0.01) (Figure 1F), signifying Sal reduced CCl₄-induced liver fibrosis.

Considering the defensive effect of Sal on liver injury and fibrosis, Sal’s influence on hepatocyte apoptosis was further studied by TUNEL staining. As indicated in Figure 2A, the apoptotic hepatocytes in the Model group were significantly amplified compared with those in the control group. Apoptotic cells in the Sal group as compared with the Model group were observed to be considerably reduced, indicating Sal’s efficacy in alleviating CCl₄-induced hepatocyte apoptosis.

Sal’s efficiency in expressing classic proteins in the mitochondrial apoptosis pathway and the role of the JNK signaling pathway in CCl₄-induced liver fibrosis were also investigated. Higher levels of cleaved caspase 3, cleaved PARP and Bax were expressed in the Model group, while Bcl-2 was lowered. Sal therapy was detected to reverse these changes and upregulate the ratio of Bcl-2 to Bax (p < 0.05 or p < 0.01) (Figure 2B). As displayed in Figure 2B, CCl₄ supported JNK phosphorylation in liver tissues. Sal therapy was recorded to downregulate the enhanced expression of p-JNK in model mice (p < 0.05) (Figure 2B). These findings indicate Sal considerably reduced mitochondria-mediated hepatocytes apoptosis by inhibiting JNK phosphorylation.

Sal's antifibrosis efficacy in relation to the regulation of SphK/S1P/S1PR signaling pathway was studied by examining the expression levels of SphK, S1P, and S1PRs. The protein and mRNA levels of SphK1 were detected to be upregulated, and SphK2 protein and mRNA expression downregulated in the liver tissues of the Model group as compared with those in the control group (p < 0.05 or p < 0.01) (Figures 3A,C). Sal was found to inhibit these effects (p < 0.01) (Figures 3A,C). Moreover, a high S1P level was observed in the model group (p < 0.01) (Figure 3B), and Sal was found to lower its level.

In addition, CCl₄ was also detected to regulate S1PRs, the receptors of S1P. Notably, it also amplified the expression of S1PR1 and S1PR3 but subdued that of S1PR2. Sal substantially eradicated the effects of S1PRs triggered by CCl₄, whether stimulating or hindering (Figure 3C).

Serum exosomes from mice and humans were successfully isolated in this research, and their characteristics were analyzed by electron microscopy (Figure 4A), while the expression of markers of exosomes, TSG101 and CD9, was identified by Western blot (Figure 4B). SphK1 was noted to be highly expressed in serum exosomes from liver cirrhosis patients, which encouraged the migration of LX-2 (p < 0.01) (Figures 4C,D). A similar remarkably high expression of SphK1 in the serum exosomes derived from mice with liver fibrosis was recorded (p < 0.01) (Figure 4E). Moreover, Sal treatment was detected to downregulate the expression of SphK1 in serum exosomes (p < 0.05) (Figure 4E). As theorized, the serum exosomes in the mice with liver fibrosis were also found to encourage the migration of JS 1, and the serum exosomes of the Sal group to subdue the migration of JS 1 (p < 0.01) (Figure 4F).

Serum-derived exosomes contain too many contents, not limited to a certain organ or even a certain type of cell. Therefore, it is very difficult to trace whether the components in serum exosomes originate from a certain cell. Considering the special anatomical position of LSEC and HSC and the essential role of the interaction between LSECs and HSC in the process of liver fibrosis (Deleve, 2015), we cultured the primary LSEC and detected its expression of SphK1, trying to prove the source of SphK1. The freshly isolated LSECs were small and spherical. During the in vitro culture, the LSECs were observed to gradually stretch out and adopt a long spindle shape (Figure 5A). Figures 5B,C present isolated LSECs phagocytosed Dil-Ac-LDL and expressed CD31, both of which are features of LSECs. Primary LSECs were also isolated from liver fibrosis model mice prompted by CCl_4, which expressed considerably higher SphK1 than that in the Wild mice (p < 0.01) (Figure 5D).

High levels of SphK1 were detected in primary mouse LSECs of liver fibrosis mice. Although the effect of exosomal SphK1 was tried to be studied on LSECs and HSC, the exosomes could not be derived owing to the inadequate number of primary LSECs. Thus, SK-HEP-1 cell lines that function similarly to LSECs were used (Cogger et al., 2008). Overexpression SphK1, SK-HEP-1, was established, and exosomes were isolated from the culture medium to study how exosomal SphK1 influenced the function of LX-2. As displayed in Figures 6A,B, substantially protein and mRNA expression of SphK1 was detected in SK-HEP-1 cells transfected with the lentivirus carrying SphK1 as compared with empty vector-transfected SK-HEP-1 cells (p < 0.05 or p < 0.01) (Figures 6A,B).

As predicted, the exosomes from the culture medium of SphK⁺-SK-HEP-1 cells released significant quantities of SphK1. Moreover, upregulation of FN was recorded in the exosomes of SphK⁺-SK-HEP-1 cells (p < 0.05 or p < 0.01) (Figures 6D,E). LX-2 cells were incubated with exosomes to explore the influence of exosomal SphK1 on the function of LX-2. As displayed in Figure 8F and G, the exosomal SphK1 caused the migration of LX-2 and the Col I level in LX-2, which could be reversed by Sal (p < 0.05 or p < 0.01) (Figures 6F,G). Earlier studies have evidenced AKT phosphorylation to support HSC activation, while exosomal SphK1 was found to encourage AKT activation, and Sal reduces this effect in this research (p < 0.05 or p < 0.01) (Figure 6G). Thus, Sal was theorized to lessen LX-2 migration and activation induced by exosomal SphK1 by hindering AKT activation.