Function Of ACSL1 In HSC/Liver Fibrosis And Its Mechanism

Objective:Liver fibrosis is a pathological state in the liver subject to certain stimuli and injury,and is characterized by excessive deposition of extracellular matrix(ECM)in the liver during repair regeneration after liver injury.The key cell leading to the liver fibrosis is hepatic stellate cell(HSC),which can be activated to transform into myofibroblast(-like)cells when stimulated by factors such as inflammation and liver cell death.Recent studies have shown that changes in lipid metabolism of HSC are very important for HSC activation.Acyl-co A synthetase long-chain 1(ACSL1)is the most highly expressed isoform in the ACSL family in liver tissue,and it is the main regulator of lipid metabolism in liver tissue.The previous work of our group has suggested that ACSL1 may inhibit liver fibrosis,but its mechanism is still unclear.Firstly,this study detected the ACSL1 expression in different clinical human liver tissue samples and analyzed the relationship between ACSL1 and HSC activation/hepatic fibrosis in vitro human HSC(LX-2 cell line).The biological role of ACSL1 in HSC activation/fibrosis was clarified in vitro experiments.Secondly,through targeted lipidomics,the changes in HSC lipid metabolism after overexpression of ACSL1 were studied and the differentially expressed lipid metabolites in HSC after ACSL1 overexpression were screened out,then by subsequent experiments the effect of differential metabolites on HSC activation/liver fibrosis was further verified,and the possible regulatory mechanism that ACSL1 affects the lipid metabolism in HSC cells and thus affects fibrosis was explained.On this basis above,it is found that the inflammatory pathway regulated by nuclear factor kappa-B(NF-?B)has a significant impact on liver fibrosis through reading a large number of literature reports,and it was found that NF-?B can bind to the ACSL1 promoter fragment through bioinformatics technology analysis and screening.Therefore,further experiments were carried out to explore the combination of NF-?B and ACSL1 and the related mechanism of NF-?B affecting liver fibrosis through the regulation of ACSL1.Methods:First,the expression of ACSL1 in liver fibrosis and other liver tissues was clarified by detecting the expression of ACSL1 in several kinds of clinical liver tissue samples;the expression of ACSL1 was verified in human hepatic stellate cell line LX-2 in vitro,and the biological function of ACSL1 on HSC/fibrosis was clarified.ACSL1 overexpression and ACSL1 interference lentivirus were constructed based on the sequence information of ACSL1.After the virus packaging was completed,the LX-2 cells were infected and the ACSL1 overexpression or ACSL1 interference stable strain transfected cells were obtained to further verify the function of ACSL1;Further,the Ed U proliferation experiment and Annexin V-APC single staining apoptosis assay were used to analyze the effect of ACSL1 on cell proliferation and apoptosis of HSC.Then,by using the constructed ACSL1 overexpression lentiviral stable strain and the corresponding control,the targeted quantitative lipidomics analysis of liquid chromatography and mass spectrometry(LCMS/MS)was performed to analyze the effects of ACSL1 in HSC on changes in HSC lipid content and on liver fibrosis-related functions;through the analysis of the changes in HSC lipid metabolism after overexpression of ACSL1,and further interventions on these lipid metabolism differences,it is clear that its effects on HSC/fibrosis influences.Finally,after predicting that NF-?B is a transcription factor upstream of ACSL1 through PROMO,and then conduct chromatin immunoprecipitation(Ch IP)experiments to verify that the transcription factor NF-?B p65 in HSC can bind to the promoter fragment of ACSL1;we clarified the regulation of ACSL1/NF-?B p65 by transforming growth factor beta-1(TGF-?1)pathway in LX-2,verified the relationship between NF-?B p65 and ACSL1 expression,and detected the influence of NF-?B pathway on the occurrence and development of liver fibrosis.Results :(1)The results of immunohistochemistry showed that the expression of ACSL1 in HSC decreased with the activation of HSC in human liver tissue samples;In the HSC in vitro experiment,after stimulation with TGF-?1,compared with the blank group,liver fibrosis related indicators collagen ?(type ? collagen alpha 1,Col1a1)and ? smooth muscle actin(?-smooth muscle actin,?-SMA)increased while the expression of ACSL1 decreased,and the expression of Col1a1 increased with the increase of the concentration of TGF-?1,while the expression of ACSL1 decreased with the increase of the concentration;(2)ACSL1 overexpressing lentivirus was obtained,puromycin-containing medium adds to infect cells for screening,and the successful selection of stable transfected strains can be used for subsequent experiments.ACSL1 interference lentivirus was obtained,the knockdown efficiency of ACSL1 was detected by q RT-PCR,and the KD1 infected cells with the highest knockdown efficiency were selected for subsequent experiments such as the functional tests and the downstream liver fibrosis index detections;(3)Overexpress ACSL1 and thus inhibit HSC collagen synthesis,and knockdown ACSL1 and thus promote HSC collagen synthesis;(4)Knockdown of ACSL1 increased the cell proliferation rate,and overexpression of ACSL1 increased the proportion of apoptosis;(5)After overexpression of ACSL1 in the LX-2 cell line,the targeted quantitative lipidomics analysis of liquid chromatography and mass spectrometry(LC-MS/MS)showed that among all lipid groups,the number of triglyceride(TG),which is the main component of HSC lipid droplets,is the largest;there is no significant change in the lipid composition of HSC after transfection of ACSL1 overexpression lentivirus;(6)After overexpression of ACSL1,the content of free fatty acid(FFA)and TG increased to varying degrees relative to the control group(the only exception is arachidonic acid<AA,one of the lipids in FFA > decreased);Through PCA analysis,compared with the control group,the lipid profiles in the HSC of the two transfected virus groups were significantly changed;the lipid metabolism data was corrected by OPLS-DA,and the the S-plot of OPLS-DA was used to observe the distribution of differential lipid metabolites between the control virus(CON)group and the ACSL1overexpression(OE)group;(7)After screening for differential metabolites,we found that the TG difference in HSC is the largest after overexpression of ACSL1,and TG may be closely related to the steady state of HSC;(8)After ACSL1 is overexpressed,when the degree of unsaturation is 0(saturated fatty acid),the lipid content is highest,and the increase in lipid content with less unsaturation may make HSC inner lipids more stable;after overexpression of ACSL1,the lipid peroxidation level in HSC is inhibited,further suggesting that ACSL1 inhibits the occurrence and development of fibrosis may be related to the lipid peroxidation content in HSC;(9)Soy phosphatidylcholine(SPC)in HSC can inhibit the expression of liver fibrosis related indicators ?-SMA and affect the content of Col1a1;while AA can promote liver fibrosis related indicators Col1a1,type ? collagen(type ? collagen alpha 1,Col3a1)expression;(10)NF-?B p65 can directly bind to the ACSL1 promoter region;(11)TGF-?1 pathway can inhibit ACSL1 and promote NF-?B p65;(12)In HSC,NF-?B p65 can inhibit the expression of ACSL1 protein,and the NF-?B pathway can promote the occurrence and development of liver fibrosis.Conclusions:(1)Clinical liver tissue sample detection and in vitro cell experiments found that: ACSL1 has the function of inhibiting HSC proliferation/suppressing liver fibrosis;(2)ACSL1 can affect the process of liver fibrosis by affecting the changes in lipid metabolism in HSC;(3)The NF-?B pathway in HSC can inhibit the expression of ACSL1 and promote the occurrence and development of liver fibrosis and this pathway may be regulated by TGF-?1.