Studies On The Anti-hypertensive Effects Of Dihydroartemisinin On Fibrotic Portal Hypertension And The Underlying Mechanisms

BackgroundPortal hypertension is a frequent pathological symptom occurring especially in hepatic fibrosis and cirrhosis.Current paradigms indicate that inhibition of hepatic stellate cell（HSC）activation and contraction is anticipated to be an attractive therapeutic strategy,because activated HSC dominantly facilitates an increase in intrahepatic vein pressure through secreting extracellular matrix and contracting.Previous studies have shown that dihydroartemisinin（DHA）is a potential antifibrotic natural product by inducing HSC apoptosis,whereas the role of DHA in regulating HSC contraction and the mechanisms involved remain a riddle.Besides,the effects of DHA on fibrosis-related portal hypertension still requires clarification.Recent studies have emphasized on the importance of farnesoid X receptor（FXR）and sphingosine-1-phosphate receptor 2（S1PR2）in controlling cell contractility and portal pressure.This study was aimed to investigate the therapeutic effects of DHA on fibrotic portal hypertension and elucidate the underlying mechanisms.Our discoveries made DHA a potential candidate for portal hypertension and provided novel insights into the mechanistic studies on the antifibrotic and antihypertensive effects of DHA.Methods1.Clinical studiesClinical liver samples from patients with liver cancer（n = 4）were randomly selected from Nanjing Drum Tower Hospital.Immunofluroscene double staining was performed to evaluate the expression of FXR in HSC in cancerous and adjacent tissues.2.In vitro studiesHuman LX-2 cells are immortalized HSC used in this study.Gel contraction assay and fluorescence analysis of cytoskeleton were used to evaluate the contractility of HSC.Intracellular calcium(Ca²⁺)concentration was measured using Ca²⁺ fluorescence probe.Immunofluorescence staining,quantitative real-time polymerase chain reaction,and western blot were performed to quantify the expression of target genes in HSC.3.In vivo studiesForty-eight male Sprague-Dawley rats were randomly divided into 6 groups（8 rats/group）and received corresponding treatment:vehicle,CCl₄,CCl₄ DHA,CCl₄+obeticholic acid（OCA）,CCl₄ + DHA + OCA,CCl₄ + DHA + Z-guggulsterone（ZGGS）.Hematoxylin-eosin staining and scanning electron microscope were performed to observe the microstructure of rat livers.Immunohistochemical staining was performed to measure the contents of proinflammatory cytokines in rat livers.Sirius red staining and Masson’s trichrome staining were performed to qualitatively assess the contents of collagen fibers in rat livers.The contents of hydroxyproline in liver tissues were quantificationally measured by a commercial kit.Activities of serum aspertate aminotransferase,alanine aminotransferase,alkaline phosphatase,and lactic dehydrogenase were detected using commercial assay kits.The levels of interferon-gamma,tumor necrosis factor-alpha,interleukin-6,hyaluronic acid,laminin,procollagen type III,collagen type IV,and transforming growth factor-beta in serum were determined by corresponding enzyme-linked immuno sorbent assay kits.Measurement of hepatic portal pressure and mean arterial pressure was performed under pentobarbital anesthesia by cannulation of portal vein and carotid artery.Immunofluorescence staining,quantitative real-time polymerase chain reaction,and western blot were performed to quantify the expression of target genes in rat livers.ResultsThe expression of FXR in HSC was significantly reduced in human liver tumor samplescompared with adjacent tissues.The in vitro data showed that DHA strongly induced the mRNA and protein expression of FXR in LX-2 cells in a dose-and time-dependent manner,and inhibited HSC activation,implying a conceivable impact of DHA on HSC contraction.The gel contraction assays and fluorescence staining of actin cytoskeleton verified that DHA dose-dependently limited contraction of collagen lattices and reorganization of actin stress fibers in LX-2 cells.DHA also decreased the phosphorylation of myosin light chain that is responsible for the contractile force of HSCs.Further gain-or loss-of-function analyses exhibited a FXR-dependent mechanism of inhibiting HSC contraction by DHA.Subsequent work revealed that inhibition of both Ca²⁺-dependent and Ca²⁺-sensitization signaling transductions contributed to DHA-induced HSC relaxation.Deeper investigations revealed that DHA decreased S1PR2 expression by modulating FXR activation and FXR/S1PR2 axis mediated the inhibitory effects of DHA on HSC contraction.The in vivo data showed that a decrease in FXR expression in CCl₄-administrated rat HSC but an increase in that in DHA-treated rat HSC.Serum diagnostics and morphological analyses consistently indicated that DHA exhibited hepatoprotective effects on CCl₄-induced liver injury.DHA also reduced CCl₄-caused inflammatory mediator expression and inflammatory cell infiltration.These improvements were further enhanced by OCA but weakened by ZGGS.Noteworthily,DHA,analogous to OCA,significantly lowered portal vein pressure and suppressed fibrogenesis.Mechanistically,inhibition of HSC activation and contraction was found as a cellular basis for DHA to relieve portal hypertension.ConclusionIn this study,data obtained from clinical liver samples showed that FXR expression in HSC was negatively correlated with the degrees of HSC activation.Our in vivo data showed that activation of FXR was a prerequisite for DHA to ameliorate CCl₄-induced liver injury and inflammation.Portal hypertension was also lowered by DHA,which might be associated with alleviated fibrogenesis and inhibited HSC contraction in a FXR activation-dependent approach.Further in vitro mechanistic study revealed that DHA limited HSC stellation by regulating FXR/S1PR2 signaling axis and further influencing Ca²⁺-dependent and Ca²⁺-sensitization pathways.Our studies provided experimental evidence for drug targeting FXR for the management of portal hypertension.