PTEN Suppresses The Activation Of Hepatic Stellate Cells Induced By Endothelin—1 By Downregulating Endothelin B Receptor Via Negatively Regulating PI3K/AKT Signaling

Background:Liver fibrosis is a reiterated process of wound-healing and scar-repairing following various types of chronic liver injury, which is characterized by the imbalance between the production and deposition of extracellular matrix (ECM) that leads to the excessive deposition of ECM and then to liver fibrosis ultimately. The progression of liver fibrosis is reversible, while advanced liver fibrosis inevitably leads to cirrhosis, tumors and even liver failure without appropriate intervention. Liver fibrosis is resulted from the interaction of various cells and cytokines, and among those that hepatic stellate cells (HSCs) were confirmed to be the most important cell type that iniate liver fibrosis and whose abnormal activation and proliferation is the key invent in the progression of liver fibrosis. Under normal circumstance, HSCs keep themselves in quiescent status and the main function is for storing Vitamin-A, which is called Ito cell as well. When liver is in case of damages, HSCs get activated following variety of inflammatory stimuli and trans-differentiate into proliferating myofibroblast-like cells that lose their vitamin-A droplet and produce excessive ECM, therefore lead to liver fibrosis. The process of HSCs activation is regulated by various substance and signaling pathway, while the specific mechanisms involved has not been well elucidated.Endothelin-1 (ET- 1) is the most potent peptide that regulates vasoconstriction, which plays important roles in the regulatory process of vascular tone in normal individuals. ET-1 is also closely related with the occurrence and progression of many disease which includes the promoting of cell growth and proliferation, wound healing and fibrogenesis. In recent years, studies about ET-1 on the progression of liver fibrosis, especially on HSCs activation have been carried out extensively. In normal liver tissue, hepatic sinusoidal endothelial cells and HSCs are major producing cells, when in case of liver injury, the majority of ET- 1 synthesis shifts to HSCs and initiate the activating process by binding endothelin receptors (ETR) on HSCs. ET -1 start-ups the activation and proliferation of HSCs via binding endothelin-1 receptors on HSCs, namely ETAR and ETBR, which promotes DNA synthesis, increasing of collagen and proteoglycan, hyperplasia of fibrous tissue and accelerated injury repairing. Many researches showed that ET-1 plays important roles in the pathophysiological process of liver fibrosis, however, the mechanistic details of how ET-1 modulates HSCs activation, proliferation, and ECM production in liver fibrosis remains unclear.Phosphate and tensin homolog deleted on chromosome ten (PTEN) is a tumor suppressor gene which has dual activities of protein and lipid phosphatase, deletion or mutation of PTEN has been linked to a range of advanced cancers. PTEN was found in several human tumors includes primary liver cancer, while recently, studies about PTEN have been shifted from liver tumor field into some benign liver disease and PTEN has been reported in literatures to be closely related with liver fibrosis. Studies showed that PTEN expressed little in fibrotic liver tissue and decreased with the progression of liver fibrosis, further more, the expression of PTEN was negatively correlated with the activation and proliferation of HSCs in vitro. Therefore, regulating the expression of PTEN in HSCs may affect the activation and proliferation of HSCs, and intervent the progression of liver fibrosis.In conclusion,ET-1 can initiate the preocess of HSCs activation while PTEN inhibits the process, whether there were some reciprocity between ET-1 and PTEN on the activation of HSCs and the specific mechanism involved has not be elucidated clearly.Aims:In order to explore deeply the effect of ET-1 and PTEN on the activation of HSCs, the present study aimed at the effect produced by PTEN-ovexpressed HSC-T6 when exposed to exogenous ET-1 and selected LY294002, the specific inhibitor of PI3K/AKT signaling to simulate the blocking effect of PTEN on PI3K/AKT, then explored whether overexpression of PTEN could inhibit ET-1-induced HSCs activation by downregulating ETBR expression via negatively regulating PI3K/AKT signaling.Methods:①、Experiment groupsThe present study was performed according to the following groups:1, group P:PTEN transfected HSC-T6+ET-1; 2, group V:empty vector transfected HSC-T6 without encoding PTEN+ET-1; 3, group PBS:cells in control group treat with PBS+ET-1; 4, group L+V:empty vector transfected HSC-T6 without encoding PTEN+ET-1+LY294002; 5, group L+PBS:cells in control group treat with PBS +ET-1+LY294002.②、Cell cultureRat HSC-T6 cell line was purchased from Guangdong Academy of Sciences and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum,100 U/ml penicillin G sodium salt, and 100 U/ml streptomycin sulfate at 37℃ and 5% CO2.③、Transient transfectionHSC-T6 cells were seeded in a 6-well plate at a density of 2×105 cells per well 24h before transfection. When 70%-80% confluence was attained, cells were transfected with a vector encoding PTEN (100 nM) or an empty control vector using Lipofectamine 2000 according to the manufacturer’s instructions; as an additional control, cells were separately treated with phosphate-buffered saline (PBS). The culture medium was replaced 6 h later. After being transfected stably, cells were exposed to 10-7mol/L ET-1 and 50uM LY294002 were then added in group L+V and L+PBS treated 12h later. After being treated with ET-1 for 48h, cells were collected for protein and mRNA assays.④、Western blot analysisα-SMA, ETBR, and ETAR protein expression levels were evaluated by western blotting. Cells treated with ET-1 for 48h were collected and lysed on ice for 30 min in radioimmunoprecipitation assay buffer containing 1% Nonidet P-40,0.5% sodium deoxycholate,0.1% sodium dodecyl sulfate, and protease inhibitors, followed by centrifugation at 13,000×g for 15 min. The supernatant was collected and protein concentration was determined using a bicinchoninic acid assay kit (Boster Biological Technology, Wuhan, China). Equal amounts of protein were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 1 × PBS containing 5% nonfat dry milk and 0.2% Tween 20 for 1 h at room temperature, then washed three times with washing buffer (0.1% Tween-20 and 1 mM Tris-HCl, pH 7.5) for 10 min and incubated overnight at 4℃ with primary antibodies against α-SMA (1:1000), ETBR (1:1500), ETAR (1:1500) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000) (Shanghai Sangon Biotech, Shanghai, China). After three 10-min washes with washing buffer, the membrane was incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:4000) (Fude Biotech). After washing, bands were detected by enhanced chemiluminescence (Millipore) and scanned with an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA). Results were shown as the ratio of the integrated absorbance values of the target protein relative to GAPDH.⑤、Quantitative reverse transcription (qRT)-PCRTotal RNA was extracted from cells treated with ET-1 for 48h using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and 1μg was used to synthesize cDNA. qRT-PCR was performed in a 20-μl reaction containing 2× Fast SYBR Green Master Mix, forward and reverse primer, the cDNA template, and dH2O on an LightCycler 480ⅡSystem PCR.β-actin was used as an internal control. The forward and reverse primer sequences were as follows:ETAR,5’-TCT GAT GAC CTC GGT CCC-3’and 5’-GTT CAT GCT GTC CTT ATG GCT-3’; ETBR,5’-AAT TAC GAT GGA CTA CAA AGG AAG TTA-3 and 5’-GCA AGC AGA AAT AGA AAC TGA ATA GC-3’; and β-actin,5’-TCA CCC ACA CTG TGC CCA TCT ACG A-3’ and 5’-CAG CGG AAC CGC TCA TTG CCA ATG G-3’.Statistical analysisData were expressed as mean±standard error. Statistical analysis was performed with vSPSS v.21.0 software. Differences between groups were compared by one-way analysis of variance (One-way ANOVA). A p<0.05 was considered statistically significant.Results:1、The expression of each index after been treated with exogenous ET-1①、α-MA expression in each group after been treated with exogenous ET-1When HSC-T6 in each group were exposed to exogenous 10-7mol/L ET-1, western blot showed that relative expression of α-SMA in group P was (0.35±0.01), which was significantly lower than those in group V and PBS (0.46±0.01,0.46±0.01, repectively p<0.05).②、ETBR expression in each group after been treated with exogenous ET-1When HSC-T6 in each group were exposed to exogenous 10-7mol/L ET-1, western blot showed that relative expression of ETBR protein in group P was (0.42±0.01),which was significantly lower than those in group V and PBS (0.85±0.01,0.83±0.01, respectively p<0.05). qRT-PCR revealed that relative ETBR mRNA expression in group P was (0.63±0.06),which was significantly lower than those in group V and PBS(1.61±0.13,1.50±0.03,respectively p<0.05).③、ETAR expression in each group after been treated with exogenous ET-1When HSC-T6 in each group were exposed to exogenous 10-7mol/L ET-1, western blot showed that relative expression of ETAR protein in group P was (0.22±0.01),there was no significant difference with those in group V and PBS (0.22±0.01,0.22±0.01, respectively p>0.05). qRT-PCR revealed that relative ETAR mRNA expression in group P was (0.46±0.01),and there was no significant difference with those in group V and PBS(0.46±0.01,0.46±0.01,respectively p>0.05).2、The expression of each index after been treated with LY294002①、α-SMA expression in each group after been treated with LY294002When HSC-T6 in group L+V and L+PBS were treated with LY294002, western blot showed that relative expression of α-SMA in group L+V and L+PBS were (0.34±0.01,0.34±0.01) that were significantly lower than that in group V and PBS (respectively, p<0.05), but there was no significant difference with that in group P (respectively, p>0.05).②、ETBR expression in each group after been treated with LY294002When HSC-T6 in group L+V and L+PBS were treated with LY294002,western blot showed that relative expression of ETBR in group L+V and L+PBS were (0.43±0.01,0.44±0.01) that were significantly lower than that in group V and PBS (respectively,p<0.05), but there was no significant difference with that in group P (respectively, p>0.05). qRT-PCR revealed that relative ETBR mRNA expression in group L+V and L+PBS were (0.65±0.02,0.64±0.01) that were significantly lower than that in group V and PBS (respectively, p<0.05), but there was no significant difference with that in group P (respectively, p>0.05).③、ETAR expression in each group after been treated with LY294002When HSC-T6 in group L+V and L+PBS were treated with LY294002,western blot showed that relative expression of ETAR in group L+V and L+PBS were (0.22±0.02,0.23±0.01) that there were no significant difference with in group P, V and PBS (respectively, p>0.05). qRT-PCR revealed that relative ETAR mRNA expression in group L+V and L+PBS were (0.45±0.02,0.46±0.01) that there were no significant difference with in group P, V and PBS (respectively, p>0.05) as well.Conclusion:PTEN suppresses ET-1-induced HSCs activation by down-regulating ETBR instead of ETAR via negatively regulating PI3K/AKT signaling.