The Study On The Effect And Mechanism Of CPhGs Liposome Against Liver Fibrosis

Objective:Construct and characterize CPhGs liposomes.Investigate the release behavior in vitro and pharmacokinetics in vivo of CPhGs and CPhGs liposomes.The rat model of immune hepatic fibrosis induced by bovine serum albumin was used to observe the effect and mechanism of CPhGs liposome on immune hepatic fibrosis in rats.On the basis of observing the effects of CPhGs liposomes on the proliferation,apoptosis,cycle and collagen secretion of hepatic stellate cells,further observing is processed on the effects of CPhGs liposomes on HSCs proliferation stimulated by rrPDGF-BB.The effects of CPhGs liposomes on MAPK and FAK/PI3K/Akt signal transduction pathways were discussed at the cellular and molecular levels so as to reveal their action targets and elucidate their molecular mechanisms.Methods:?1?CPhGs liposomes were prepared by film dispersion primary entrapment method,thin film dispersionsecondary entrapment method,reverse evaporation method and vacuum freezing drying film dispersionsecondary entrapment method.The morphology of liposome was observed by transmission electron microscope,the particle size and Zeta potential were measured by Malvern laser particle size analyzer,the drug entrapment efficiency and drug loading rate were measured by and HPLC method,and the preparation method was evaluated.?2?HPLC was used to detect and clarify the drug release rule of CPhGs liposomes in vitro.LC-MS was used to determine the time course of echinacetin in CPhGs liposomes in rat plasma.The pharmacokinetic characteristics were analyzed and compared with CPhGs raw materials.?3?The rat model of immune hepatic fibrosis was established by using BSA.The effects of CPhGs liposome and CPhGs on immune hepatic fibrosis induced by BSA were observed.The changes of serological indexes,liver histopathology,immunohistochemistry,ECM protein,MAPK pathway and inflammatory protein expression were analyzed to evaluate the effect of CPhGs on liver fibrosis and its possible molecular mechanism.?4?The effects of CPhGs liposome on proliferation,apoptosis,cell cycle,cell viability and cytotoxicity of HSCs were observed in vitro.The effect of CPhGs on liver fibrosis was expounded at the cytological level,and the effect of CPhGs liposome on PDGF-BB/FAK/PI3K/Akt signal pathway was studied.The target of CPhGs liposome against liver fibrosis was discussed,and its mechanism was clarified at molecular level.Results:?1?The average entrapment efficiency of three different batches of CPhGs liposomes prepared by the film dispersion once encapsulation method is 28.55%,and the drug loading rate is 2.73%.The average entrapment efficiency of the three different batches of CPhGs liposomes prepared by the thin film dispersionsecondary encapsulation method is 38.46%,and the drug loading rate is 3.71%.The average entrapment efficiency of three different batches of CPhGs liposomes prepared by reverse evaporation method was 33.88%,and the drug loading rate was 3.28%.The entrapment efficiency and drug loading rate of CPhGs liposomes prepared by thin film dispersion twice entrapment method was both the highest.On the basis of CPhGs liposome prepared by film dispersion twice entrapment method,CPhGs liposome freeze-dried powder was prepared by vacuum freeze-drying method.The appearance of CPhGs liposome freeze-dried powder was good after resolution,and the particle size and entrapment efficiency of CPhGs liposomes changed little before and after freeze-drying,indicating that the nanoparticles were stable.?2?The results of drug release in vitro showed that the most suitable models for CPhGs liposomes and CPhGs were Higuchi and first-order kinetic equation,while the study of CPhGs liposomes group did not show any sudden release effect.The related parameters of pharmacokinetics in rats showed that Cmax in blank liposome group and CPhGs liposome group increased significantly after administration of CPhGs liposome?P<0.05?.The plasma concentration of echinacoside in rats after administration of CPhGs liposome was much higher than that of raw drug?35.95±3.95 vs 27.14±0.93 ng/ml?.After intragastric administration of CPhGs,the concentration of echinoside in rats was lower,CL was 2.07±0.39 ml/h/kg,t_1/2 was significantly extended?CPhGs liposome group 5.33±0.43h vs CPhGs group 2.07±0.61h,P<0.05?,and MRT was significantly extended?CPhGs liposome group 5.87±0.32h vs CPhGs group 2.80±0.47h,P<0.05?,Tmax increased significantly?CPhGs liposome group 1.60±0.55h vs CPhGs group 0.60±0.22h,P<0.05?.The area under the drug-time curve of CPhGs liposome group in rats within 24 hours was significantly higher than that of CPhGs group,which was 273.30%of that of CPhGs group.?3?Both CPhGs liposome group and CPhGs raw drug group could decrease the liver and spleen index of hepatic fibrosis model rats,and the liver and spleen index of CPhGs liposome group was significantly lower than that of CPhGs group.In addition,in the same dose of CPhGs targeted liposome group,the serum ALT,AST,ALP,TBIL,TBA,?-GT,TGF-?,LN,HA,PCIII,IV-C and HYP in liver tissue were significantly lower than those in CPhGs group,and the liver pathological condition of rats with hepatic fibrosis was also improved.The results of immunohistochemistry and WB experiments showed that CPhGs targeted liposomes could significantly reduce the protein expression of ECM marker molecules?-SMA and Collagen?.Compared with model group,CPhGs group and blank liposome group,MAPK signal pathway related proteins p-ERK 1/2,p-JNK 1/3and p-P38,and inflammation related proteins NF-?B,AP-1,Cox-2 and HMGB1 were down-regulated and I?B?was up-regulated in liver fibrosis rats.?4?The inhibition rate of CPhGs liposome on HSCs proliferation increased with the increasing of drug concentration,and the 24-hour IC₅₀ value of 42.535?g/ml.CPhGs liposome?29.45,14.72,7.36?g/ml?had no obvious toxic effect on HSCs.The results of flow cytometry showed that CPhGs liposome at the concentration of 29.45?g/ml could induce late apoptosis in HSCs,and the proportion of late apoptotic cells was higher than that of early apoptotic cells.In the CPhGs liposome group with the concentration of 14.72?g/ml and 7.36?g/ml,the proportion of early apoptotic cells was significantly higher than that of late apoptotic cells.The results of cell cycle showed that compared with the normal control group,after HSCs were treated with different concentrations of CPhGs liposomes,the proportion of cells in G0/G1 phase increased significantly?P<0.05?,and the proportion of cells in S phase decreased significantly?P<0.05?,and the proportion of cells in G2/M phase decreased significantly?P<0.05?.Compared with rrPDGF-BB group,different concentrations of CPhGs liposomes could inhibit the proliferation of HSCs induced by rrPDGF-BB.RT-PCR results showed that compared with rrPDGF-BB group,29.45,14.72,7.36?g/ml CPhGs liposomes groups could reduce the mRNA expression of Collagen?,?-SMA and TIMP-1 and increase the expression of MMP-1 mRNA.In the activated HSCs stimulated by rrPDGF-BB,the expression levels of Collagen?and?-SMA mRNA were significantly increased.However,the mRNA expression levels of Collagen?,?-SMA and TIMP-1 were decreased by CPhGs liposomes in all three dose groups,especially in the high dose CPhGs liposome group?29.45?g/ml?.WB analysis showed that the expression of Collagen?and?-SMA protein in the model group was significantly higher than that in the normal control group?P<0.05?.Compared with the model group,the protein expression levels of Collagen?and?-SMA of CPhGs liposomes in 7.36?g/ml,14.72?g/ml and 29.45?g/ml groups decreased significantly?P<0.05?.After HSCs were treated with CPhGs liposome for 24hours,the levels of p-PI3K and p-Akt in HSCs decreased continuously.The expression of FAK protein in rrPDGF-BB stimulation+FAK gene overexpression group was significantly higher than that in rrPDGF-BB stimulation group,while there was no significant change in FAK protein expression in rrPDGF-BB stimulation+FAK gene overexpression control group.Compared with rrPDGF-BB+FAK gene overexpression group,the expression of phosphorylated PI3K and phosphorylated Akt protein in rrPDGF-BB+FAK gene overexpression control group and rrPDGF-BB+FAK gene overexpression+CPhGs liposome group decreased significantly?P<0.01?.Conclusion:the CPhGs liposome nanoparticles prepared by film dispersion twice entrapment method have the advantages of round shape,high entrapment efficiency,small particle size distribution and good stability.The results of drug release experiments in vitro show that the CPhGs liposome nanoparticles prepared in this study have sustained release effect to some extent,and significantly changed the pharmacokinetic behavior of CPhGs,slowed down the elimination time of the drug,and improved the bioavailability of the drug.CPhGs liposome can reduce liver and spleen index,improve serum liver function enzyme index,reduce serum liver fibrosis marker content,reduce hepatocyte degeneration and inflammatory cell infiltration,inhibit collagen fiber synthesis and deposition,and regulate liver fibrosis through MAPK pathway and inflammatory factor protein expression.CPhGs liposome administration could significantly reduce thesecretion of HSCs on the basis of no cytotoxicity,inhibit the proliferation of HSCs stimulated by rrPDGF-BB in a dose-dependent manner,and regulate the expression of ECM protein in HSCs stimulated by rrPDGF-BB through PDGF/FAK/PI3K/Akt pathway.The results provide a scientific basis for the application of CPhGs liposome nanoparticles in anti-hepatic fibrosis.