The Study On Anti-Liver Fibrosis Activity Of Benzoquinone And Indazole Derivatives

Benzoquinone is a kind of important organic compounds,which distributed widely in animals,plants and microorganisms.Because of the conjugate diketone moiety in the structure that shows reversible two-electron transfer coupled with proton transfer to form a redox cycle which becomes the active center of redox reaction,Show a certain biological activity.Indazole derivatives was a kind of rare nitrogen heterocyclic compounds in nature which has many bioactivities and play an irreplaceable role in the field of chemical biology.But the anti-liver fibrosis mechanisms of both have not to be well reported.Hepatic fibrosis is caused by chronic inflammation,which leads to the abnormal hyperplasia of connective tissue in the liver and makes the extracellular matrix appear excessive deposition.It is a compensation response in the process of self-repair after liver injury.Therefore,it is very significant to take advantage of the structure characteristics of the compounds to discover the biological activity which has not been developed.Based on this,in this paper,we take the anti-liver fibrosis activity of benzoquinone derivatives study.Methods:In this paper,through two aspects which include electrochemical methods and vitro biological activity test of TQ(Thymoquinone),tBu-Q(2-tert-Butyl-1,4-benzoquinone),Dime-Q(2,5-Dimethyl-1,4-benzoquinone)and Ph-Q(Phenyl-1,4-benzoquinone)to make an anti-liver fibrosis research.In vitro,we investigated the anti-liver fibrosis activity on the basic of lipopolysaccharide(LPS)-activated human hepatic stellate cells line(LX-2),which have characteristics with inflammation and fibrosis.(1)Cyclic voltammetry was employed to measure the redox characteristics of benzoquinone derivatives;(2)Through CCK-8 assay to investigate cell viability on activated LX-2 cell of benzoquinone derivatives,and choose the appropriate concentration used for subsequent biological experiment;(3)Using the flow cytometry to detect the change of ROS level,mitochondrial membrane potential and the cell apoptosis;(4)On the molecular level,investigate the protein expression of CD 14 and TLR4,PI3K/AKt,ERK1/2 and LKB1-AMPK which associated with liver fibrosis,Bcl-2/Bax,Caspase3,XIAP,FILP protein related to apoptosis,Caspasel,IL-1 beta,LC3 correlated to inflammation and autophagy,a-SMA(alpha smooth muscle protein),Collagen I,TIMP-1(matrix metalloproteinases inhibitor 1)which served as liver fibrosis iconic protein.At the same time,we detected the effects on activated LX-2 cell of Indazole derivatives by CCK-8 assay.Using the flow cytometry to test the change of the membrane potential,cycle arrest and apoptosis;At last,through western blot experiments to investigate the proteins expression of Bcl-2 family,Caspase3,PDGF-BB and liver fibrosis iconic protein of alpha SMA.Four kinds of benzoquinone derivatives have shown the following activity of anti-liver fibrosis:Through two-step single electron reduction process to become Semiquinone and hydroquinone structure respectively,thus to play a role of antioxidant;The four types of Benzoquinone derivatives inhibit the viability of activated LX-2 cells,the IC50 were 69.99 μM,61.39 μM,53.88 μM,41.78 μM respectively;Eliminating the ROS in the cells which elevated by LPS and causing the decrease of mitochondrial membrane potential at the same time;Four compounds induce the activated LX-2 cells apoptosis effectively,inhibit the protein expression involved in liver fibrosis,and induce mitochondrial autophagy to reduce the damage of mitochondria to eliminate excess ROS which suppress the activation of NLRP3 so as to reduce inflammation reactions in the mean while.Significantly reduce the expression of liver fibrosis iconic protein;Four kinds of indazole derivatives inhibited the viability of activated LX-2 cells,induced apoptosis accompany with the decreased of mitochondrial membrane potential,caused cell G1-phase cycles arrest,increased Bax/Bcl-2 ratio,activated Caspase3 protein expression,reduced the PDGF-BB and inhibited the expression of alpha SMA significantly.Conclusions:The four types of Benzoquinone derivatives could play a role in anti-liver fibrosis through three pathways:Through reacting with the redox system in cell to become oxidation species,which eliminated the ROS in the activated LX-2 cells effectively and inhibited liver fibrosis process;By inducing autophagy not only remove the activation of hepatic stellate cells but also reduce the damaged mitochondria,eliminate excess ROS to suppress the activation of NLRP3 in order to reducing inflammation;They could induce cell apoptosis at the same time.Indazole derivatives regulated the mitochondria death signals to induced LX-2 cell apoptosis,which remove the effector cells of the liver fibrosis;They could down-regulate the expression of promote mitosis protein and cause the cell cycle arrest in G1 phase in order to hinder the process of liver fibrosis.Finally decreased the expression of a-SMA significantly which showed a certain activity of anti-liver fibrosis.