Study On The Active Ingredients From Cichorium Glandulosum Against Hepatic Fibrosis And Their Mechanism Of Action

Objective:To obtain the active part and ingredients of anti-hepatic fibrosis in cichoriumglandulosum root,and to explore the mechanism of anti-hepatic fibrosis of sesquiterpene and total flavonoids(TFCG)from C.glandulosum root through activity trace in vivo and invitro.Method:Compounds from C.glandulosumroot were obtained and identified with the aid of column chromatography,Pre-HPLC and 2D-NMR technology.Firstly,the protective effect of 95%ethanol extract of C.glandulosumroot(CG-I)on the immunological liver injury induced by concanavalin(Con A)in mice was investigated.Sixty Konmin mice were randomly divided into 6 groups(n=10):high-,moderate-or low-doseCG-I,positive drug,control and model groups.Mice in CG-I and positive drug groups were orally treated with CG-I and diammoniumgly cyrrhizinate,and mice in control and model groups were gavaged with normal salineonce a day for 10 days.One hour after the end of gavage on day 10(except for the normal group),mice were treated with a caudal vein injection of Con A.Serum indices,inflammatory factors,liver tissue homogenate indices and liver tissue HE staining of mice in each group were detected.The CG-I was extracted to obtain the abstract of petroleum ether(CG-V),ethyl acetate(CG-VI)and N-butyl alcohol(CG-VII),which were used to the intragastric administration of rats with liver fiber model induced by thioacetamide(TAA).Sixty Wistar rats,fifty-fifty for male and female,were divided into 6 groups:CG-V(15mg·kg-1),CG-VI(3mg·kg-1),CG-?(6mg·kg-1),silibinin capsules(8mg·kg-1),normaland model groups.With the exception of normal group,mice in the rest groups were performed with an intraperitoneal injection of TAA(100mg·kg-1)twice a week.Since the 5th week of modeling,mice in the normal and model groups were orally administered with distilled water,and mice in the other groups were gavaged with corresponding drugs once a day for 8 weeks(12 weeks in total).The condition of liver,spleen index,serum indices,pathological test,protein expression and cell apoptosis in hepatic tissue were investigated.The inhibitoryeffect of 3 separated sesquiterpene lactone compounds on TGF-?1-stimulated proliferation of HSC-T6 and their effect on gene expressions on 24/48h were investigated using MTT and RT-PCR assays.TFCG were separated and purified from C.glandulosumroot,and the irmechanismagainst hepatic fibrosis in rats was investigated.Except for the control group,the rats in the rest groups were firstly treated with a subcutaneous injection of 40%CCL4-vegetable oil suspension at a dose oflml·kg-1 for the first time and 0.5ml·kg-1 for the following treatment,and the rats in control group were injected with normal saline of equal volume twice a week.At the end of 4th week,the rats in medicated group were randomly divided into model,positive(silibinin),TFCG high-dose(200mg·kg-1),moderate-dose(100mg·kg-1)and low-dose(50mg·kg-1)groups.The rats in medicated group were gavaged from the start of 5th week for 13 weeks in total.The invivo liver and spleen coefficient,serum indices,pathological section,immunohistochemistry and protein expressions of rats were detected.RT-PCR assay was used to investigate the inhibitory effect of TFCG on the TGF-?1-stimulated proliferation of HSC-T6 and their effect on gene expressions on 24/48h.The effect of PERK gene slow virus modification in human normal hepatic cell line L02 on the apoptosis mediated by endoplasmic reticulum stress were tentatively explored.Result:Three sesquiterpene lactone compounds were identified as11?,13-Dihydrolacurin,austricia,8-deacetylmatricin-8-O-sulfate and ?-sitosterol.The three active ingredients included a new compound,a new natural product,and an ingredientfirstly isolated from C.glandulosum root.The protective effect of CG-I on immunological liver injury in rats was described as follow.Compared with the model group,the level of ALT,AST,GST and AKP in CG-I medication group were all significantly reduced(P<0.05 or P<0.01),and the liver and spleen coefficients were all significantly reduced(P<0.05 orP<0.01).Meanwhile,the levels of IFN-y,TNF-?,IL-1?and MDA inCG-I high-dose group were significantly reduced,and the levels of T-SOD and albumin were significantly elevated(P<0.05 or P<0.01).The results of HE staining indicated thatthe degree of live injury in each CG-I medicated group was reduced.The results of the effect of 3 abstracts on hepatic fibrosis induced by TAA in rats suggested that compared with the model group,CG-VII reduced the liver and spleen coefficients(P<0.05);CG-VIIreduced the spleen coefficient(P<0.01),and the activities of AST and ALT in the serum of rats in medicated group was significantly reduced(P<0.05 or P<0.01).The results of HE and Masson staining detection indicated that the degree of hepatic fibrosis intreatment group(CG-V and CG-VII)were significantly reduced relative to the model group.The results of immunohistochemistry assay suggested that compared with model group,CG-V and CG-VII significantly reduced the expressions of FN?Smard3 and TGF-?1 and the apoptosis index(P<0.05 or P<0.01).The results of Western-Blot assay exhibited thatthe expressions of FN and Smard3in CG-V and CG-VII groupswere significantly reduced relative to the model group(P<0.05),and the expression of IGFBPrP1 in CG-VII group was significantly reduced relative to model group(P<0.05).The results of the effects of 3 sesquiterpene lactonecompounds on HSC-T6 gene expression indicated that theyshoweda significant inhibitoryeffect on the expressions of Smad2,Smad3,PPP5C and TGF-?1,and theyexhibitedan extremely significant inhibitoryeffect on the expressions of above genes at 24hour(P<0.01),but the inhibitory effect at 48 hour was not significantly different.All of the 3 sesquiterpene lactonecompoundssignificantly up-regulated the gene expression of Smad7 at 48h(P<0.05 or P<0.01),while,only some concentration of Austricin and 8-deacetylmatricarin-8-O-sulfate up-regulated the gene expression of Smad7at24h(P<0.05 or P<0.01).Meanwhile,the effects of 3 sesquiterpene lactonecompounds on Smad7gene expressionshowed a time-dependent manner..The technological conditions of TFCG extraction were:80?,extraction with 70%ethyl alcohol with a solid-liquid ratio of 1:15 for 2 h,two times.The purification conditions of AB-8 were:upper sample concentration 3.Omg·ml-1,flow rate 4 BV·h-1,5BV column volume,absorption rate>80%;50%ethanol elution,flow rate 2BV·h-1 and desorption rate>90%within 2BV column volume.The content of TFCG was 50.82±0.37(n=3).The results of the preventive effect of TFCG on hepatic fibrosis induced by CCL4 in rats indicated thatthe liver and spleen coefficients of TFCG and silibinin groups were both reduced to some extent(P<0.05)relative to model group.Meanwhile,compared with the model group,the levels of ALT,AST,AKP and LDH inserum of rats in all the medicated groups were all significantly reduced(P<0.05 or P<0.01),the y-GTlevel inthe TFCG high-and low-dose groups and silibinin group was significantly different(P<0.01),theserum Hyp level in TFCG and silibinin groups was significant different(P<0.05 or P<0.01),the level of albumin in theTFCG high-and low-dose groups and positive drug group was significantly increased(P<0.05),the level of MDA inTFCG high-dose group was significantly reduced(P<0.01).The result of pathological examination suggestedthat the hepatic fiber of rats in various doses of TFCG groups was significantly improved.The result of immunohistochemistry suggested that all various doses of TFCG groups significantly reduced the protein expressions of TGF-?1,Smad3,TLR4 and a-SMA(P<0.01 or P<0.05),and up-regulated the expression of Smad7(P<0.01).The result of Western-Blot exhibited that compared with the model group,the expression of TGF-?1inTFCG moderate-and high-dose groups and positive group were significantly reduced(P<0.05),and the expression of Smad7 was significantly enhanced(P<0.05).The result of RT-PCR suggested that as for the medicated group,the inhibitory effect of TFCG on expressions of TGF-?1 and PPP5CmRNA at 24 h was better than that at48h.As to the up-regulation of Smad7gene,the effect at 48h was better than that at 24h.Meanwhile,the effects of TFCG on Smad7gene expression showed a time-dependent manner.However,theeffect of TFCG onTGF-?1,PPP5C,Smad2 and Smad3 genes expressionsdidn't exhibited a time-dependent manner.TFCG moderate concentration group exhibiteda significant effect on gene expression(P<0.01).The Human protein kinase endoplasmic reticulum kinase(PERK)lentiviral vector was tentatively built and wrapped with the lentivirus particles,and the cell apoptosis of L02 was investigated using Western-Blot,flow cytometry and other technologies.Conclusion:Through activity trace,the mechanism of sesquiterpene and TFCGagainst hepatic fibrosis has been explored,and 3 lead compounds are obtained.The sesquiterpenoids down-regulate the expressions of FN,IGFBPrP1,Smad3 and TGF-?1 proteinsin the hepatic tissue,inhibit the expression of TGF-?1,Smad2,Smad3,PPP5CmRNA in HSC-T6,up-regulate the expression of Smad7mRNA,and inhibit the activation and proliferation of HSC-T6 and effectively block the signal transduction pathway of TGF-?/Smad.TFCG inhibits the expressions of TGF-?1,Smad3,TLR4 and a-SMA in the hepatic tissue,up-regulates the expression of Smad7 protein,showsa significantly inhibitoryeffect on the expression of TGF-?1,Smad2,Smad3 and PPP5CmRNA in the activated HSC-T6.Meanwhile,TFCG can extremely significantly up-regulate the mRNA expression of Smad7 gene,effectively block the signal transduction pathway of TGF-?/Smad,and exhibitsa significant inhibitory effect on the protein and gene of a-SMA,TLR4 and PPP5C.It was tentativethat during an endoplasmic reticulum stress,LV-PERK lentivirus can promote the proliferation and apoptosis of hepatic cell line L02.