Study On The Mechanisms Of Sesame Active Ingredients Regulating FXR/SHP/LXR Signals To Ameliorate Hepatic Fibrosis

Objective: Hepatic fibrosis is a pathological process of abnormal connective tissue proliferation,accompanied by collagen synthesis and excessive deposition of extracellular matrix(ECM).The main causes include alcohol,drugs,metabolic disorders,viral infections,immune hepatitis,and cholestasis.Liver cirrhosis and liver cancer caused by hepatic fibrosis are the main causes of morbidity and mortality worldwide.The incidence of liver disease is increasing year by year.China is a major country with liver disease,seriously damaging people health and increasing the burden on society.Current treatment methods include drugs,fibrogenic markers and other technologies.However,it is necessary to combine a lot of clinical evaluations to ensure its effect.Western medicine has been clinically reported to improve viral hepatitis,but it still lacks specificity for fibrous connective tissue hyperplasia.Traditional Chinese medicine components or active ingredients show obvious advantages in anti-hepatic fibrosis.Our research team discovered the sesame,a natural plant,has antiinflammatory and antioxidant pharmacological activities.It was speculated that sesame may attenuate liver disease.Thus,the main active ingredients,sesamin and sesamol,were selected to verify whether they can ameliorate hepatic fibrosis.The mechanism of sesamin and sesamol against hepatic fibrosis is still unclear,so it restricts the development and application of new drugs.Therefore,our study carried out the sesamin and sesamol against hepatic fibrosis in vivo and in vitro,and analyzed the mechanism of anti-hepatic fibrosis.The study provided pharmacological reference for clinical treatment of hepatic fibrosis,and theory for the development of new drugs in basis.Sesamin(SES)and sesamol(DER)are natural lignan compounds with various pharmacological activities such as anti-inflammatory and antioxidant.However,the of effect of SES and DER on ameliorating hepatic fibrosis is still unclear.Therefore,this topic mainly studies the related mechanisms of SES and DER to attenuate hepatic fibrosis.Specifically: How SES regulates hepatic inflammation and fibrosis in vivo and in vitro through farnesoid X receptor(FXR)/ short heterodimer partner(SHP)signaling pathway;DER inhibits the activation of rat hepatic stellate cells(HSC-T6),regulates FXR/LXR signal crosstalk,participates in the interaction between THP-1 macrophages and human hepatic stellate cells(LX-2),and inhibit the activation of HSCs.Methods:(1)C57BL/6 mice were randomly divided into six groups: Normal group,TAA group,TAA and SES(20 mg/kg)group,TAA and SES(40 mg/kg)group,TAA and curcumin(Cur)(20 mg/kg)group and SES(40 mg/kg)group.In addition to the normal group and the single SES group,the other groups were injected with TAA intraperitoneally for 5 weeks,100 mg/kg three times a week in the first week and 200mg/kg twice a week in the second to the fifth weeks.In addition,in the durgs administration group,mice were treated with SES(20,40 mg/kg)or Cur(20 mg/kg)by oral gavage for 28 consecutive days from the second week.At the same time,normal group and TAA group were given the same volume of normal saline for 4 weeks.After the experiment,we detected the serum biochemical indicators aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels.Then,Sirius red staining,Masson staining and H&E staining were used to examine the histopathological changes.The expressions of the corresponding antibodies were detected by immunohistochemistry and immunofluorescence staining.Finally,Western blot and quantitative Reverse Transcription PCR(q RT-PCR)were used to detect the expressions of related hepatic fibrosis indicators and inflammatory factors.In vitro,HSCs were activated by TGF-β,and then treated with SES(3.125,6.25,12.5 μM),FXR agonist GW4064(2 μM)or FXR inhibitor gugglusterone(50 μM)for 2h.The expressions of FXR,SHP,α-SMA,nucleotide-binding domain-(NOD-)like receptor protein 3(NLRP3)、interleukin-1β(IL-1β)、interleukin-1α(IL-1α)and other proteins were detected by immunofluorescence、Western blot and q RT-PCR.Specific gene blocking FXR(si FXR)and SHP(si SHP)were used to transfect HSCs,and the expression of related proteins was detected by western blot and q RT-PCR.(2)C57BL/6 mice were randomly divided into six groups: Normal group、TAA group、TAA and DER(10 mg/kg)group、TAA and DER(20 mg/kg)group、TAA and Cur(20 mg/kg)group and DER(20 mg/kg)group.TAA administration method is the same as SES.In addition,the mice in the drugs treatment group were treated with DER(10and 20 mg/kg)by oral gavage for 28 consecutive days from the second week.At the same time,normal group and TAA group were given the same volume of normal saline for 4 weeks.In the autophagy-induced hepatic fibrosis model,mice were randomly divided into 5 groups: normal group,TAA group,TAA and DER(20 mg/kg)group,TAA and RA(2 mg/kg)group,TAA and 3-Methyladenine(3-Methyladenine,3-MA)(10 mg/kg)group.The administration method of the TAA group was the same as SES group.From 2 to 5 weeks,except for the normal group and the TAA group,the other two groups were injected with RA or 3-MA 3 times a week.After the experiment,the serum biochemical indicators and histopathological changes were detected.Western blotting and fluorescent quantitative reverse transcription polymerase chain reaction(Reverse Transcription PCR,RT-PCR),q RT-PCR methods were used to determine the expression of fibrosis indicators and inflammatory factors.In the in vitro model,HSCs were first activated with TGF-β,and then treated with DER(3.125,6.25,12.5 μM)to detect FXR,LXRα/β,and microtubule-associated protein light chain 3α/β(Microtubule-associated protein light chain 3α/ β,MAPLC3α/β)and ubiquitin-binding protein autophagy receptor(Sequestosome 1,SQSTM1/P62)and other related proteins.After activating HSCs with transforming growth factor-β(TGF-β),RA and 3-MA,the expression of related proteins was detected after DER treatment.In addition,THP-1cells were stimulated with LPS(0-100 ng/ml),and we collected the supernatant lipopolysaccharide(LPS).LX-2 cells were stimulated combined with RA.After incubation with DER or 3-MA,the expressions of autophagy proteins MAPLC3α/β,p62,α-SMA and inflammation related proteins were detected by Western blotting,RTPCR and cell fluorescence.HSCs were treated with FXR agonist GW4064 、LXR agonist GW3965 and DER to detect the expressions of autophagy 、 α-SMA and inflammation related proteins with RT-PCR and cell fluorescence.Results:(1)In the model of hepatic fibrosis induced by TAA,SES significantly reduced the elevation of ALT and AST levels induced by TAA,and ameliorated the histopathological changes.SES also significantly inhibited α-SMA,type I collagen(collagen-I),tissue inhibitor of metalloproteinase-1(TIMP-1)/matrix metalloproteinase-13(matrix Metalloproteinase-13,MMP13)and other fibrosis-related marker proteins and inflammatory cytokine infiltration,and up-regulate the expression of FXR and SHP.In vitro results show that SES can significantly inhibit the expression of hepatic fibrosis marker proteins caused by TGF-β stimulation.The regulation of SES on NLRP3 and other inflammatory cytokines was confirmed by western blot,q RT-PCR and cell immunofluorescence.SES and FXR agonist GW4064 had the same effect.SES(12.5 μM)significantly increased the protein and immunofluorescence expressions of FXR and SHP,and down regulated the protein expressions of α-SMA、IL-1α and IL-6.After FXR and SHP gene silencing,SES regulated the m RNA and protein expressions of FXR、SHP、IL-1α and IL-6 by regulating the interaction between FXR and SHP.(2)DER significantly reduced the serum biochemical indicators ALT and AST levels caused by TAA,improved histopathological changes,and significantly inhibited the expression of fibrosis marker proteins and autophagy proteins.DER also downregulates the expression of inflammatory cytokines,such as cysteinyl aspartate specific proteinase-1(caspase-1)and IL-6,and inhibits the activation of NLRP3 inflammasomes.DER has the same effect as the autophagy inhibitor 3-MA,significantly down-regulating the levels of ALT and AST,and ameliorating the histopathological changes caused by TAA or RA.In vitro results showed that DER significantly inhibited the expression of fibrosis marker proteins and inflammatory cytokines in HSCs caused by TGF-β stimulation.DER treatment reversed the FXR and LXRα/β protein expression caused by TGF-β activation of HSCs.TGF-β and RA to activate HSCs,DER and 3-MA have similar functions,inhibiting the autophagy proteins and the release of inflammatory cytokines.The supernatant of THP-1 cells stimulated by LPS was collected and incubated with LX-2 cells.DER and 3-MA inhibited the expression of autophagy protein,α-SMA and inflammatory cytokines.DER also inhibited the activation of LX-2 cells induced by LPS-conditioned medium by gene blocking autophagy-related protein 7(ATG7)(si ATG7).In addition,DER with the similar functions as GW4064 or GW3965,up-regulated the expressions of FXR and LXR in LX-2 cells,and inhibited the activation of autophagy proteins and NLRP3 inflammasomes.After gene silencing FXR(si FXR)and LXR(si LXR),DER or 3-MA also inhibited the activation of LX-2 cells.Conclusion:(1)SES inhibits the activation of NLRP3 and the expressions of inflammatory cytokines by mediating the FXR-SHP signaling pathway.SES reduces the deposition of ECM,and inhibits the activation of HSCs.Through gene silencing FXR and SHP,we the verified the interaction of FXR and SHP.SES acts as an agonist of FXR,activating the expression of FXR and SHP.In addition,SES protects the liver from the TAA toxicity,prevents the accumulation of collagen and ECM,reduces the damage of inflammatory cytokines to the liver,and changes in histopathology.(2)DER participated in the interaction between LX-2 cells and THP-1 cells by targeting FXR/LXR signals.DER can inhibite the autophagy reaction of LX-2 cells,reduce the activation of NLRP3 inflammasomes,the lysis of caspase-1 and the product of inflammatory cytokine IL-1β.DER has the same functions as 3-MA,inhibited the activation of HSCs and autophagy and reduced the expression of hepatic fibrosis marker proteins caused by TAA.DER also improved histopathological changes,reduced the activation of NLRP3 inflammasomes,infiltration of inflammatory factors and hepatic fibrosis development.In addition,DER and 3-MA can inhibit liver autophagy,relieve the damage of autophagy caused by RA in the liver,reduce liver inflammation and histopathological changes.In conclusion,this study confirmed that the natural lignan compounds SES and DER participate in the FXR/SHP/LXR signal interaction.They have good effects on improving hepatic fibrosis and inhibiting inflammation,and preventing TAA and autophagy from damage to the liver.DER can improve hepatic fibrosis by regulating the crosstalk between macrophages cells and HSCs.These findings fill the gaps in the field of DER and SES in the field of anti-hepatic fibrosis.SES and DER can be used as ideal regulators to improve hepatic fibrosis,and have the unique biological activity advantages of natural medicines.In the future,we will do more applications and research in the pharmacokinetics,bioavailability,and safety evaluation of SES and DER.This will lay a good theoretical foundation for developing safe,effective and innovative anti-hepatic fibrosis drugs for clinical development.