Critical roles for liver sinusoidal endothelial cells (LSECs) in liver fibrosis have been demonstrated, while little is known regarding the underlying molecular mechanisms of drugs delivered to the LSECs. Our previous study revealed that plumbagin plays an antifibrotic role in liver fibrosis. In this study, we investigated whether plumbagin alleviates capillarization of hepatic sinusoids by downregulating endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), laminin (LN), and type IV collagen on leptin-stimulated LSECs. We found that normal LSECs had mostly open fenestrae and no organized basement membrane. Leptin-stimulated LSECs showed the formation of a continuous basement membrane with few open fenestrae, which were the features of capillarization. Expression of ET-1, VEGF, LN, and type IV collagen was enhanced in leptin-stimulated LSECs. Plumbagin was used to treat leptin-stimulated LSECs. The sizes and numbers of open fenestrae were markedly decreased, and no basement membrane production was found after plumbagin administration. Plumbagin decreased the levels of ET-1, VEGF, LN, and type IV collagen in leptin-stimulated LSECs. Plumbagin promoted downregulation of ET-1, VEGF, LN, and type IV collagen mRNA. Altogether, our data reveal that plumbagin reverses capillarization of hepatic sinusoids by downregulation of ET-1, VEGF, LN, and type IV collagen.
Inflammation, chronic viral hepatitis, and liver injury are considered to be canonical causes of hepatic fibrogenesis. Liver injury leads to loss of liver sinusoidal fenestration, which may promote fibrosis by sinusoidal thrombi [
While LSECs have large amounts of open fenestrae, they lack a continuous basement membrane [
Upregulation of endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), laminin (LN), and type IV collagen by LSECs during liver fibrosis has been reported [
In our previous study, we found a drug, plumbagin, that suppressed hepatic stellate cell (HSC) activation and contributed to the activation of HSC apoptosis during liver fibrosis
We hypothesized that plumbagin would reverse liver fibrosis in another way, through its accumulation in LSEC and subsequent amelioration of phenotype changes and dysfunction via decreasing profibrogenic factors, including ET-1 and VEGF, as well as reducing components of basement membrane LN and type IV collagen. In this study, we delivered plumbagin to leptin-stimulated LSECs. Our purpose was to illustrate that the effects of plumbagin on alleviating phenotype changes and dysfunction in the leptin-stimulated LSEC were achieved via the decreased expression of ET-1, VEGF, LN, and type IV collagen, effectively alleviating the mechanism causative of hepatic fibrosis.
Plumbagin was purchased from Sigma Chemical Company (St. Louis, MO, United States). Dimethylnitrosamine (DNM) was purchased from Dahao Chemical Company (Shantou, Guangdong, China). Dimethylsulfoxide (DMSO) was purchased from Solarbio Technology Company (Beijing, China). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO.
The experiments were performed using 8-week-old male Sprague Dawley (SD) rats, which were obtained from the Medical Laboratory Animal Center of Guangxi Medical University, China (using number SCXK-Gui-2009-0002). The body weights of the rats were 150~180 g. At least 3 animals were needed for the experiment. The animal studies were performed in the Animal Research Center at Guangxi University of Chinese Medicine (Nanning, Guangxi) and approved by the Institutional Animal Ethics Committee of Guangxi University of Chinese Medicine, China. All authors ensured that all experiments were performed in accordance with the approved guidelines and regulations. All animals were maintained under standard conditions (22°C and 12 h light/dark cycles) for further experiments.
To obtain primary LSECs, male Sprague Dawley (SD) rats were used. Isolation of primary rat LSECs was performed as published [
For maintaining LSECs phenotype after the isolation, on the one hand, the cells were cultured in DMEM containing 10% FBS and 10 ng/mL endothelial cell growth factor (ECGF, purchased from PeproTech, United States). On the other hand, primary cells seeded on coverslips coated with collagen were used for experiments and were rapidly fixed with glutaraldehyde for electron microscopy.
All authors ensured that the experiments protocols related to animals were approved by the Institutional Animal Ethics Committee of Guangxi University of Chinese Medicine. All experiments with animals were performed in accordance with the approved guidelines and regulations.
The cells were grown in six-well plates. To stimulate LSECs, 100
The cells were divided into 3 groups: (1) vehicle control group without leptin or drug; (2) leptin group, or the leptin-stimulated LSECs model group, without any drug treatment, in which 100
To investigate changes in LSEC fenestrae, we used scanning electron microscopy (SEM). Twenty-four hours after administration, all samples were prepared for SEM. The preparations were washed using phosphate-buffered solution (PBS) twice and then fixed rapidly with 2.5% glutaraldehyde for 1 hour. The preparations were washed with PBS twice, followed by 30%, 50%, 70%, and 80% ethanol at 4°C and 90%, 95%, and 100% ethanol at room temperature to dehydrate the prepared samples, all for 10 minutes. The preparations were then placed in 100% isoamyl acetate ester and dried. The preparations were placed on a metal stub. Platinum (15 nm) was used as the surface coating for the prepared samples. The samples were viewed with a VEGA3 scanning electron microscope (Hitachi S-4800, Tokyo, Japan).
The isolated LSECs were rapidly immersed in 2.5% glutaraldehyde for 1 hour and then washed in PBS three times. Then, 1% OsO4 was added for 2 hours to fix the cells. The cells were then dehydrated via a graded acetone solution. Epoxy was used to embed the cells. Ultrathin sections were then passed through copper grids, and uranyl acetate and lead citrate were added to stain the sections. Finally, the sections were imaged using a transmission electron microscope (Hitachi H-7650, Tokyo, Japan). The sections were stained using uranyl acetate and lead citrate. Copper grids were used to maintain the ultrathin size of the sections.
To investigate the expression of ET-1, VEGF, LN, and type IV collagen in LSECs, cells on coverslips were washed with PBS three times for three minutes each time and then fixed with 4% poly-formaldehyde solution (5 ml per well). After 15 minutes, the cells were washed with PBS three times. Then, the cells were incubated in 0.5% Triton X-100 containing goat serum and incubated with anti-ET-1, anti-VEGF, anti-LN, and anti-type IV collagen and the corresponding Cy3-conjugated anti-IgG antibodies. Coverslips were mounted on glass slides with anti-fluorescence quenching sealing (Southern Biotech Company) mounting medium with DAPI (Beyotime, Biotech Company, Shanghai, China) and visualized using a fluorescence inverted microscope (Olympus, Japan).
The levels of ET-1, VEGF, LN, and type IV collagen in LSECs were determined via ELISA using the corresponding assay kits (Elabscience) according to the manufacturer’s instructions. Briefly, medium was added to ELISA plates coated with anti-rat ET-1, VEGF, type IV collagen, and LN antibody (Elabscience) and incubated at 37°C for 1.5 hours. Next, the corresponding biotinylated detection antibody was added to the plates and incubated at 37°C for 1 hour. This was followed by adding of avidin-conjugated horseradish-peroxidase (HRP) to the plates. Finally, chromogenic substrate was added to the plates. An ELISA microplate reader was used to detect levels of rat ET-1, VEGF, type IV collagen, and LN antibodies.
To investigate the levels of ET-1, VEGF, LN, and type IV collagen mRNA in LSECs, quantitative RT-PCR was performed. Total RNA was extracted from LSECs using a total RNA Extraction Kit (TIANGEN BIOTECH, Beijing, China). Then, total RNA reverse transcription into cDNA was performed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). For qPCR, SYBR Green or Fluorescein qPCR Master Mix (2x) (Thermo Scientific, Waltham, USA) was used. Specific PCR primers sequences were as follows: ET-1 forward, 5′-TCCCGTGATCTTCTCTCTGC-3′, reverse, 5′-TGACCCAGATGATGTCCAGG-3′ (212 bp); VEGF forward, 5′-CGTCTACCAGCGCAGCTATTG-3′, reverse, 5′-CTCCAGGGCTTCATCATTGC-3′ (145 bp); type IV collagen forward, 5′-GGGTGATTGTGGTGGCTCTG-3′, reverse, 5′-CCTCGTGTCCCTTTCGTTCC-3′ (198 bp); LN forward, 5′-GACCCGTTCGGTTGTAAAT, reverse, 5′-GCCAGACTCCACCTCGTTA-3′ (288 bp);
Data were analyzed using SPSS statistic 16.0 software and presented as the mean ± SD. ANOVA with Bonferroni posttests were used to evaluate significant differences between groups.
In normal LSECs, there were large amounts of open fenestrae and no continuous basement membrane. In contrast, continuous basement membranes were visible in the leptin-stimulated group. Few open fenestrae were observed in the leptin-stimulated group. All of the above results indicated that leptin mediated the defenestration of LSECs and the formation of a basement membrane, as shown by scanning electron microscope and transmission electron microscopies (Figures
Plumbagin returned fenestrae loss in vitro study: the numbers and diameter of fenestrae were detected by scanning electron microscope examination. (a) Control group had large numbers of open fenestrae with 1.83–2.13 diameter. (b) There were no open fenestrae in the leptin-stimulated group. (c) Plumbagin with 2
Plumbagin reverses capillarization of hepatic sinusoid, as seen by transmission electron microscopy. LSECs were stimulated via leptin and then treated with plumbagin (2, 15
After 2 and 15
To detect the expression of ET-1, VEGF, LN, and type IV collagen in LSECs, immunofluorescence was used. ET-1 (Figure
Plumbagin decreases the levels of ET-1, VEGF, LN, and type IV collagen, as determined by immunofluorescence assay. LSECs were treated with plumbagin after stimulation with leptin. Cells treated with vehicle were used as controls. ET-1 (red) were widely observed in cytoplasm of LSECs. The proteins were immunostained using cytokeratin-19 antibody and are shown in red. Nuclei were stained using DAPI (blue). Scale bar, 20
Plumbagin decreases the levels of VEGF, as determined by immunofluorescence assay. VEGF (red) were widely observed in cytoplasm of LSECs.
Plumbagin decreases the levels of LN, as determined by immunofluorescence assay. LN (red) accumulated on cytomembrane of LSECs.
Plumbagin decreases the levels of type IV collagen, as determined by immunofluorescence assay. Type IV collagen (red) accumulated on cytomembrane of LSECs.
The ELISA results showed that leptin-induced LSECs produced large amounts of ET-1, VEGF, LN, and type IV collagen compared with the levels in the vehicle control (
Protein expression by ELISA after treatment with plumbagin. (a) Levels of ET-1, (b) levels of VEGF, (c) levels of LN, and (d) levels of type IV collagen. The data are analyzed using ANOVA with Bonferroni posttests through SPSS 16.0 software. The results are displayed in a bar graph as the mean ± SD.
We assessed ET-1, VEGF, LN, and type IV collagen mRNA expression in LSECs by qPCR and found that LSECs from the vehicle control showed lower levels. Conversely, leptin stimulation in LSECs resulted in a considerable upregulation of levels of ET-1, VEGF, LN, and type IV collagen mRNA compared to levels in the vehicle control. After plumbagin administration, LSECs expressed notably lower levels of ET-1, VEGF, LN, and type IV collagen mRNA than the plumbagin-untreated group. These effects of plumbagin were also dose dependent (Figure
qPCR analyses for ET-1, VEGF, LN, and type IV collagen mRNA in LSECs. (a) Levels of ET-1 mRNA, (b) levels of VEGF mRNA, (c) levels of LN mRNA, and (d) levels of type IV collagen mRNA. The data are analyzed using ANOVA with Bonferroni posttests through SPSS 16.0 software. The results are displayed in a bar graph as the mean ± SD.
Uncontrolled chronic liver injuries result in liver fibrosis, which is characterized by an enhanced extracellular matrix (ECM) [
Sinusoidal capillarization is characterized by the formation of a basement membrane and defenestration of LSEC. It precedes hepatic fibrogenesis and promotes fibrotic processes [
ET-1 is a potent regulator of vascular function produced in the liver via LSEC. In CCL4-induced mice liver fibrosis, ET-1 gene expression is increased [
Endothelial NO synthase (eNOS) can be stimulated by ETBR to enhance NO production [
VEGF, a key regulator of angiogenesis, mediates proliferation and migration of LSECs during the wound healing process [
LN is shown to be significantly increased in fibrotic rats and chronic hepatitis patients [
Excessive depositions of ECM proteins, containing types I, III, and IV collagens, LN, and fibronectin in the liver, are features of hepatic fibrogenesis [
In conclusion, our results reveal that leptin-stimulated LSECs present basement membrane formation due to defenestration. Upregulation of profibrogenic factors, including ET-1, VEGF, and basement membrane components (LN and type IV collagen), also occurs in the leptin-stimulated LSECs during capillarization of hepatic sinusoids. Plumbagin mitigates capillarization of hepatic sinusoids via downregulating ET-1, VEGF, LN, and type IV collagen, and this provides new insight into the signaling pathways that plumbagin can affect in LSECs. Moreover, a new therapeutic strategy for liver fibrosis may be further explored based on this study.
Plumbagin ameliorates capillarization of hepatic sinusoids by downregulating ET-1, VEGF, LN, and type IV collagen in vitro.
The authors declare that there are no financial conflicts of interest.
Guiyu Li and Yue Peng contributed equally to this study and should be considered as co-first authors.
The authors thank Guangxi University of Chinese Medicine (Guangxi, China) for providing laboratory equipment and technological support. This study was supported by the National Natural Science Foundation of China (Grant nos. 81403189, 81460628, 81660705, and 81560690) and an Innovation Research Program of Guangxi University of Chinese Medicine (Grant no. YJS201643).