Li-Ming Jin , b, , Yun-Xing Liu , b , Jin Cheng , Lin Zhou , b , Hi-Yng Xie , b ,Xio-Wen Feng , b , Hui Li , b , Yn Shen , b , Xio Xu , b , Shu-Sen Zheng , b,*

a Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310 0 03, China

b NHC Key Laboratory of Combined Multi-organ Transplantation, Hangzhou 310 0 03, China

c Department of General Surgery, Hepatobiliary and Pancreatic Surgery and Minimally Invasive Surgery, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital of Hangzhou Medical College, Hangzhou 310014, China

TotheEditor:

Hepatic ischemia-reperfusion (I/R) injury is the component of liver injury related to liver transplantation and liver surgery [ 1-3 ].The hepatic sinus microcirculation injury is a central part of hepatic I/R injury, which eventually leads to hepatocyte injury. This process is closely associated with the participation of liver nonparenchymal cells such as Kuffer cells, hepatic stellate cells, liver sinusoidal endothelial cells (LSECs), and cytokines. The activated LSECs and ischemic hepatocytes generate oxygen free radicals,promote cell adhesion, hinder hepatic sinus microcirculation, and damage the hepatocytes eventually [ 4 , 5 ]. These in turn inhibit the activation of LSECs, improving hepatic sinus microcirculation during hepatic I/R injury. Recent studies have shown that sphingosine 1-phosphate (S1P), one of the sphingolipid metabolites, has anti-apoptotic, anti-inflammatory properties and counteracts hepatic I/R injury [ 6 , 7 ]. Hence, this study was to investigate the protective mechanisms of LSECs in hepatic I/R injury through SphK1/S1P signaling.

Forty rats were randomized into four groups (n= 10 each group): sham group: at 30 min before operation, 0.5 mL saline was injected intravenously, and sham operation was performed without ischemia; I/R group: at 30 min before operation, 0.5 mL saline was injected intravenously, and the rats were subjected to 70%hepatic ischemia for 60 min; IR + phorbol-12-myristate-13-acetate(PMA) group: the saline was replaced by PMA (a SphK1 agonist,180 μg/kg), and the operation was the same as the I/R group;IR + N, N-dimethyl-D-erythro-sphingosine (DMS) group: same as PMA rats but DMS (a SphK1 blocker, 5 mg/kg) instead. All rats were sacrificed at 6 h after operation. The blood samples were collected, centrifuged, and stored at -80 °C until analysis. The hepatic specimens were fixed in 10% formaldehyde and embedded inparaffin. The experimental protocol was approved by the Animal Care Committee of Hangzhou Medical College (20180033).

Table 1 Real-time PCR primer sequences.

The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and S1P contents were measured via enzymelinked immunosorbent assay (ELISA) for assessing the degree of liver damage and sphingomyelin metabolism. Hematoxylin & eosin(HE) staining and TUNEL staining of hepatic specimens were performed for morphological observation. Real-time fluorescent quantitative polymerase chain reaction (PCR) and Western blotting were performed to detect theSphK1gene ( Table 1 ). All the values were expressed as mean ± standard deviation (SD), and were analyzed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was analyzed by Student’st-test and Chi-square analysis. APvalue of＜0.05 was considered statistically significant.

The results of the study showed that, compared with the sham group, serum ALT and AST were significantly increased at 6 h after operation in the I/R group (ALT: 24.80 ± 10.08 vs. 896.20 ± 206.80 U/L,P＜0.01; AST: 32.40 ± 12.89 vs. 10 08.0 0 ± 260.23 U/L,P＜0.01). Moreover, serum ALT and AST levels were significantly lower in the IR + PMA and IR + DMS groups than in the I/R group, but these values were not significantly different between the IR + PMA and IR + DMS groups (ALT: 621.80 ± 52.97 vs. 869.20 ± 197.43 U/L;AST: 907.80 ± 225.19 vs. 557.80 ± 154.67 U/L, allP＞0.05).

Fig. 1. The pathological changes of hepatic tissues at 6 h after reperfusion (HE staining, original magnification ×200). HE: hematoxylin & eosin.

HE staining showed that the PMA preconditioning improved hepatic I/R injury by reducing the expansion of hepatic sinus and improving the degree of damage to LSECs, while DMS preconditioning aggravated LSECs damage. The microscopic structure of the hepatic tissues showed no destruction in the sham group ( Fig. 1 A).The hepatic tissue of rats in the I/R group showed disordered structure of hepatic lobules and extremely extended hepatic sinusoids ( Fig. 1 B). Compared with the I/R group, the damage of hepatic tissues was significantly alleviated in the IR + PMA group( Fig. 1 C); and the hepatic sinuses were more obviously extended with hyperemia in the IR + DMS group ( Fig. 1 D).

TUNEL staining showed that the number of TUNEL-positive cells in rat’s liver of the I/R group were significantly increased[(28.83 ± 2.06)%,P＜0.05]. The rate of TUNEL-positive cells was further increased [(61.92 ± 7.72)%,P＜0.01] in the IR + DMS group.In contrast, a significant decrease in the apoptotic rate was observed in the IR + PMA group [(13.59 ± 3.02)%,P＜0.05] ( Fig. 2 ).

Compared with the sham group, ELISA showed that the serum S1P concentration was increased significantly in the I/R group(15.83 ± 3.14 vs. 3.46 ± 0.27 ng/mL,P＜0.05), PMA treatment further increased S1P (36.01 ± 10.13 vs. 15.83 ± 3.14 ng/mL,P＜0.05),DMS treatment decreased S1P to 4.71 ± 0.42 ng/mL ( Fig. 3 A). Realtime PCR showed that SphK1 mRNA in the I/R group was increased significantly (P＜0.01). The level of SphK1 mRNA was significantly higher in the IR + PMA group than in the I/R group (P＜0.01).Compared with the I/R group, the SphK1 mRNA was significantly lower in the IR + DMS group (P＜0.01; Fig. 3 B). Western blotting( Fig. 3 C) showed that the expressions of sphingosine-1-phosphate receptor (S1PR), intercellular adhesion molecule-1 (ICAM-1) and myeloperoxidase (MPO) in hepatic tissues were significantly increased in the I/R group compared with the sham group (S1PR:1.65 ± 0.12 vs. 1.02 ± 0.07,P＜0.05; ICAM-1: 3.21 ± 0.39 vs.1.07 ± 0.10,P＜0.05; and MPO: 2.51 ± 0.20 vs. 1.01 ± 0.05,P＜0.05; Fig. 3 D). The expression of S1PR in the IR + PMA group was the highest among the four groups and was significantly higher than that in the I/R group (2.50 ± 0.09 vs. 1.65 ± 0.12,P＜0.05).Compared with the I/R group, the expression of ICAM-1 and MPO in hepatic tissues was significantly reduced in the IR + PMA group(ICAM-1: 1.52 ± 0.26 vs. 3.21 ± 0.39,P＜0.05; MPO: 1.09 ± 0.13 vs. 2.51 ± 0.20,P＜0.05). Compared with the IR group, the protein expression of S1PR in the IR + DMS group was reduced (S1PR:1.31 ± 0.09 vs. 1.65 ± 0.12,P＜0.05), and both ICAM-1 and MPO were increased (ICAM-1: 5.92 ± 0.22 vs. 3.21 ± 0.39,P＜0.05;MPO: 5.21 ± 0.43 vs. 2.51 ± 0.20,P＜0.05).

The ultrastructure of LSECs, hepatocytes and sinusoids at 6 h after reperfusion was observed under transmission electron microscope. The shape and structure of LSECs, hepatocytes and sinusoids were normal in the sham group. In the I/R group, the cells were slightly enlarged, while the deformed cells were scattered in the hepatic tissues, the intercellular space was obviously enlarged, and even expanded. Compared with the I/R group, the changes in cellular and subcellular structural changes were close to normal in the IR + PMA group, while the cells showed obviously pyknosis and damaged subcellular structure in the DMS group ( Fig. 4 ).

Fig. 2. TUNEL-positive cells were measured in hepatic tissues in different groups.

LSECs from the I/R group were swollen and deformed obviously, showing larger intercellular space. After PMA preconditioning, the hepatic sinusoidal space returned to its normal size, and the damage of LSECs structures was alleviated, while the pyknosis of LSECs and related damage were more severe in the IR + DMS group. The change in LSECs might be an important factor that affects the hepatic sinus microcirculation. Meanwhile, the SphK1/S1P protects hepatic tissue from hepatic I/R injury and this might be associated with hepatic sinus microcirculation. It has been proven that SphK1 regulates the adhesion of actin to LSECs and leukocytes. Binding to specific receptor S1PR on the surface of endothelial cells, S1P activates GTPase Rac, which is a signaling molecule in its downstream pathways, then leads to actin movement, promotes cytoskeleton rearrangement, and finally forms a tight F-actin ring around the cells [8] . In our study, S1P has significantly reduced the damage to endothelial cells, and this might be related to actin translocation, promoting cytoskeleton rearrangement in LSECs.

In conclusion, the present study demonstrated that SphK1/S1P signaling was involved during hepatic I/R injury and has protective effects on hepatic sinusoid microcirculation in rats.

Acknowledgments

None.

CRediT authorship contribution statement

Li-Ming Jin : Investigation, Data curation, Funding acquisition,Writing - original draft. Yuan-Xing Liu : Data curation. Jian Cheng :Formal analysis, Funding acquisition. Lin Zhou : Conceptualization,Resources. Hai-Yang Xie : Formal analysis. Xiao-Wen Feng : Data curation, Methodology. Hui Li : Data curation. Yan Shen : Formal analysis. Xiao Xu : Conceptualization, Writing - review & editing.Shu-Sen Zheng : Conceptualization, Supervision.

Funding

This study was supported by grants from the Scientific Research Foundation of Traditional Chinese Medicine of Zhejiang Province(2015ZA085), Scientific Research Project of Zhejiang Provincial Health Commission (2021448467), and the Basic Public Welfare Research Project of Zhejiang Province (LGF20H030011).

Ethical approval

The experimental protocol was approved by the Animal Care Committee of Hangzhou Medical College (20180033).

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

Fig. 3. The expression of the relative proteins and transcription of SphK1mRNA in different groups. A : Serum S1P concentration in different groups; B : the transcription level of SphK1mRNA in different groups; C : representative Western blotting and ( D ) their densitometric analyses showing the expression of S1PR, ICAM-1 and MPO in the liver.GAPDH was used as an internal control. *: P ＜ 0.05; **: P ＜ 0.01.

Fig. 4. The ultrastructural changes of LSECs, hepatocytes and sinusoids at 6 h after reperfusion in different groups under transmission electron microscope (original magnification ×60 0 0).