Study Of The Effect And Mechanism Of Adiponectin On Hepatic Stellate Cells Contraction Induced By Endothelin-1

BackgroundDuring the chronic liver injury, Hepatic Stellate Cells (HSCs) undergo a processof transdifferentiation from a resting phenotype to a myofibroblast-like phenotypecharacterized by expression of fibroblastic cell markers such as a-smooth muscle actin(a-SMA). Activated HSCs produce increased amounts of ECM components thatcontribute significantly to the fibrotic changes in cirrhosis. In addition, activatedHSCs gain contractile phenotype and increasing evidence suggests that the contractileforce generated by HSCs contributes to the regulation of sinusoidal blood flow and thedevelopment of portal hypertension. A number of vasoactive molecules can triggercontractile response in HSCs with endothelin-1(ET-1) being the most potentconstrictor.ET-1induced contraction of HSCs,which has been thought to be linked toSinusoidal contraction and increased intrahepatic vascular resistance. Therefore,regulating the contraction of HSCs will currently regarded as a new approach ofcirrhotic portal hypertension therapy. Adiponectin is an adipocyte-specificadipocytokine. Recent studies have found that adiponectin can prevent thedevelopment of hepatic fibrosis.Our previous research showed that adiponectin couldsignificantly increase the inducible nitric oxide synthase (iNOS) gene and proteinexpression and nitric oxide (NO) synthesis by activating AMPK pathway, which caninhibit HSCs proliferation and transdifferentiation. However, if adiponectin couldaffect contraction of HSCs and if the mechanism was related to the expression of ET-1and the interaction with ET-1, the revelation of the changing mechanism in theintrahepatic portal hypertension vascular resistance has important clinical significance.Research in this area has not been reported.ObjectiveActivated HSC-T6were used as object of study, to observe the contractility of HSCs under conditions of adiponectin and ET-1together, to detection of what effecton genes and the biological characteristics of ET-1under the treatment of exogenousadiponectin to HSCs, and to investigate the impact of adiponectin on HSCs contractileresponse following stimulation by ET-1and its possible mechanism, in order toprovide a new theoretical basis for adiponectin reducing portal hypertension.Methods(1) To observe the effects of adiponectin and ET-1on HSC-T6contraction：Collected HSCs-T6with logarithmic growth phase, cell suspensions were layered ontop of formed Collagen lattices, the cells were given relevant drug interventionrespectively after synchronization, then divided into six groups: A. control group; B.adiponectin(0.5μg/mL) treated group; C.ET-1(10nmol/L)treated group; D. adiponectin(0.25μg/mL) and ET-1(10nmol/L) co-treated group; E. adiponectin(0.5μg/mL) andET-1(10nmol/L) co-treated group; F.adiponectin(0.5μg/mL) and ET-1(10nmol/L) andL-NAME(5mmol/L) co-treated group. The effect of different drugs on cell contractionwas measured by monitoring the change in lattices area over24hours after latticeswere detached.(2) To study the expression of ET-1in HSC-T6and the effect of adiponectin onits expression：The expression of ET-1in HSC-T6was detected by qRT-PCR andWestern blot. The expression of ET-1in the supernatants of HSC-T6was detected byELISA. Cells were grouped as follows: A. control group; B. adiponectin (0.5μg/mL)treated group; C. adiponectin (0.5μg/mL) and L-NAME (5mmol/L) co-treated group.(3) To investigate the underlying mechanism of adiponectin on HSC-T6contraction induced by ET-1: The expression of iNOS in HSC-T6was detected byqRT-PCR and Western blot. Western blot was used to detect the phosphorylationactivity of AMPK and Akt in cells. Experimental groups: A: control group; B: ET-1(10nmol/L) treated group; C: Adiponectin(0.5μg/mL) and ET-1(10nmol/L) co-treatedgroup; D: Adiponectin(1μg/mL) and ET-1(10nmol/L) co-treated group; E:Adiponectin (2μg/mL) and ET-1(10nmol/L) co-treated group.Results (1) HSCs contraction was determined by collagen gel contraction assay：Compared with the control group, ET-1(10nmol/L) could cause HSC-T6contractionsignificantly (P<0.01). Adiponectin could inhibit contraction of HSC-T6induced byET-1,and in a dose-dependent manner. Compared to that of ET-1-treated HSC-T6,treatment with adiponectin(0.5μg/mL) could inhibit the ET-1-mediated(10nmol/L)contraction significantly in fully activated HSC-T6(P<0.01). Compared to that ofET-1-treated(10nmol/L) HSC-T6, treatment with L-NAME (5mmol/L) could partiallyattenuate the inhibition of adiponectin-treated(0.5μg/mL) HSCs responded to theET-1-induced contraction (P<0.05).(2) The expression of ET-1mRNA in HSC-T6was detected by qRT-PCR:Compared to that of untreated HSCs there was a significant decrease in the mRNAexpression level of ET-1during the process of adiponectin-treated(0.5μg/mL)(P<0.05); In spite of a decrease in the mRNA expression level of ET-1in theadiponectin (0.5μg/mL) and L-NAME (5mmol/L) co-treated group, there was nosignificant difference compared with the control group(P＞0.05). The results ofexpression of ET-1protein in HSC-T6by Western blot were consistent with themRNA expression above. The protein expression of ET-1in the supernatants ofHSC-T6showed a similar result by ELISA.(3) The expression of iNOS mRNA in HSC-T6was detected by qRT-PCR: Theexpression of iNOS mRNA was found in HSC-T6, and there was a significantdecrease in the mRNA expression level of iNOS during the process of ET-1-treated,under the treatment with different concentration adiponectin, the mRNA expressionlevel of iNOS in HSC-T6increased compared with ET-1treated group, and showeddose-dependent tendency. The results of expression of iNOS protein in HSC-T6byWestern blot method were consistent with the mRNA expression above. The proteinexpression level of AMPK、p-AMPK、Akt、p-Akt in HSC-T6was evaluated byWestern blot method: there was a slight expression of p-AMPK in control group, theexpression of p-AMPK was nearly negative in ET-1-treated group. Under thetreatment with different concentration adiponectin, the expression of p-AMPK in HSC-T6increased compared with ET-1-reated group, and in a dose-dependentmanner(P<0.01). The total AMPK between experimental groups showed nosignificant difference. The expression of p-Akt was significantly increased inET-1-treated group, while the expression of p-Akt in adiponectin and ET-1co-treatedgroup was decreased compared with ET-1-treated group, and with the increasingconcentration of adiponectin the expression of p-Akt had a gradually reducing trend.The total Akt between experimental groups showed no significant difference.Conclusion(1) Adiponectin treatment could inhibit the ET-1-induced contraction in fullyactivated HSCs.(2) Adiponectin treatment could inhibit the synthesis of ET-1in mRNA andprotein level in activated HSCs, and inhibit HSCs from releasing ET-1.(3) Adiponectin negatively regulated the contractile response of HSCs to ET-1possibly via activation of AMPK, increasing in NO production and inhibition of Aktpathway.