Studies On The Effect Of Curcumol-regulated LSEC Autophagy And Angiogenesis In Anti-liver Fibrosis And The Molecular Mechanism

ObjectiveThe occurrence and development of liver fibrosis is accompanied by the activation of hepatic stellate cell(HSC)in the resting state,the accumulation of extracellular matrix(ECM)and even the dysfunction of liver cells.Liver fibrosis is the early stage of liver cirrhosis.Unlike liver cirrhosis,liver fibrosis is a reversible process.Liver fibrosis and liver cirrhosis will cause more than 1 million deaths worldwide each year,so it is urgent to suppress the occurrence and development of liver fibrosis.Angiogenesis refers to the process of forming new blood vessels on the basis of original blood vessels.In recent years,the role of pathological angiogenesis in the occurrence and development of liver fibrosis has been continuously revealed.Studies have shown that when liver fibrosis occurs,there is a serious disorder of hepatic sinusoidal vascular structure.At this time,pathological angiogenesis is abnormally intensified,leading to local hypoxia in liver tissue and increasing liver cell damage,further aggravating liver fibrosis.Liver sinusoidal endothelial cell(LSEC),angiogenesis effector cells,directly participates in the process of liver angiogenesis and sinusoidal homeostasis.Therefore,inhibiting the pathological angiogenesis of the liver driven by LSEC may slow down the progression of liver fibrosis.Autophagy is a dynamic process that degrades waste and damaged organelles or other intracellular components through lysosomes,and the degraded small molecules are reused by cells to maintain cell homeostasis.Recent investigations have found that autophagy is a mechanism that regulates the angiogenesis process of many diseases,and autophagy can regulate angiogenesis in liver cancer.However,in the research progress of liver fibrosis,the relationship between autophagy and angiogenesis is not clarifiedNatural active ingredients have the advantages of high efficiency,non-toxicity and multiple targets in the treatment of liver fibrosis.The preliminary research results of our laboratory have found that curcumol,an active ingredient in the ginger family plant,has good anti-liver fibrosis effects.In addition,the laboratory Another research basis shows that curcumol can inhibit LSEC pathological angiogenesis.Here,we further explore the specific molecular mechanism of curcumol in regulating LSEC autophagy and angiogenesis.Methods1.Cell researchThe primary rat LSEC is selected as the cell experiment object of this paper.Western blot,Real-time PCR,and immunofluorescence techniques were used to detect LSEC autophagy levels and the expression of angiogenesis-related factors.Cell scratch and Transwell cell migration tests were used to detect the migration ability of LSEC.Matrigel angiogenesis tests were used to detect the angiogenesis ability of LSEC in vitro.The morphology of autophagosomes and mitochondria in LSEC was observed by electron transmission electron microscope.Co-immunoprecipitation and laser confocal experiments were used to detect the binding and co-localization of transcription factor KLF5 and autophagy substrate p62 in LSEC.DCFH-DA probe detects ROS.Mito Sox Red probe detects LSEC mitochondrial superoxide level,JC-1 staining detects mitochondrial membrane potential in LSEC,and kits detect SOD,GSH and MDA levels in LSEC supernatant.2.Animal researchA mouse liver fibrosis model induced by carbon tetrachloride(CCl4)was established and ATG5 and KLF5 gene overexpressing mice were constructed.Curcumol(30mg/kg)was given for treatment,and the specific groups were:normal control,CCl4,CCl4+Curcumol,CCl4+curcumol+control carrier,CCl4+curcumol+ATG5,CCl4+curcumol+KLF5,CCl4+ATG5,CCl4+KLF5.Hematoxylin and eosin(HE)staining was used to detect liver damage in mice,Masson staining and Sirius red staining were used to detect collagen deposition levels,and ELISA kits were used to detect liver damage indicators in the serum of mice.The levels of four indicators of liver fibrosis,immunohistochemistry to detect the expression of key autophagy molecules and angiogenesis-related factors in liver tissues,and immunofluorescence double staining to detect the co-localization of CD31 and related target genes.Results1.It is confirmed again that curcumol can inhibit LSEC angiogenesis in vitro.In addition,curcumol can reduce the level of LSEC autophagy,and curcumol can inhibit LSEC angiogenesis by regulating cell autophagy.2.Further exploration found that the transcription factor KLF5 played an important role in the process of LSEC angiogenesis.Importantly,overexpression of ATG5,a key autophagy factor,will weaken the inhibitory effect of curcumol on KLF5.Through a deeper mechanism exploration,it is found that curcumol could promote the binding and co-localization of autophagy substrate p62 and KLF5,thereby reducing KLF5 expression.3.Curcumol reduces the content of mitochondrial ROS in LSEC.In addition,it is found that the ROS/ERK signaling pathway plays an important role in the angiogenesis process of LSEC,and finally shows that curcumol regulates the expression of KLF5 through the ROS/ERK signaling pathway.4.The liver tissue morphology analysis and the detection of serological indicators show that curcumol can improve the liver fibrosis induced by CCl4 and the pathological angiogenesis mediated by LSEC in mice.Immunohistochemistry and immunofluorescence double staining technology experiments show that in the occurrence and development of rat liver fibrosis,curcumol exerts anti-LSEC angiogenesis effect by regulating the expression of KLF5 mediated by autophagy.ConclusionThis study found for the first time that curcumol can inhibit the pathological angiogenesis process of LSEC by regulating the level of autophagy.Further studies have found that curcumol can reduce the expression level of the transcription factor KLF5.It was also explored that curcumol-inhibited autophagy led to the accumulation of autophagy substrate p62,which in turn promoted the binding of p62 and KLF5 to promote the proteasome degradation of KLF5.In addition,curcumol can regulate the expression of KLF5 through the ROS/ERK signaling pathway.This paper once again confirms that curcumol can inhibit pathological angiogenesis in the process of liver fibrosis,and deeply reveals that autophagy may become a mechanism for regulating angiogenesis,providing a reliable experimental basis for curcumol to become a clinical drug for the treatment of liver fibrosis.