| Objective: Hepatic fibrosis is a repair reaction of caused by variedpathogenic factors., is also a common pathological basis and an inevitablestage of chronic liver diseases developing into hepatic cirrhosis. There is ahigh incidence rate of liver diseases in our country, and chronic hepatitis Band C are commonly encountered and seriously influence the people's meanlife and life quality,so it has become a quite important countermeasure oftreating chronic liver diseases to stop or reverse the liver fibrosis progress.themain characteristics of hepatic disease are the increased over synthesis andirregular deposition of extracellular matrix(ECM).it is believed that hepaticstellate cells (HSC) are the original cell of liver extracellular matrixformation.The activation and proliferation of hepatic stellate cells(HSC) arekey links of liver fibrosis,which can produce mass of ECM by expression ofvarious signal-transducing protein.As we know so far, TGF-βl is the mostpotent fibrogenesis cytokine with certain effect on promoting ECM inHSCs.the bioactive signals of TGF-βl were transferred into cells to stimulatethe activation and proliferation of HSCs when TGF-βl combined with thereceptor of TGF-β(TGF-β receptor,TβR) on HSCs and the TGF-βl-TβRsignaling complex was formed.the signaling complex could phosphorylate thedownstream signaling proteins–smads, which could transfer the signals intothe nuclei. With the clarification of pathogenesis of HF,specialists in the fieldof chronic hepatic diseases are focusing on the study of prevention,treatmentand reversing of HF.several western medicines had made some progress in thisfield,such as,(peroxisome proliferators-activatedreceptor gamma,PPARγ)ligand-rosiglitazone. PPARγis a transcription factor of nuclear receptor,whichis activated by ligands.some researches indicated that;quiescent HSC expressed PPARγ,but its expression in activated HSC is obviouslydiminished.the reduction of PPARγ expression is closely correlated withactivation of HSC.it is now considered that:as PPARγligand, rosiglitazoneincreases collagenase activity,reduces collagen deposition and inhibits HSCproliferation.On the other hand,the treatments with traditional Chinesemedicine(TCM)based on the principle of blood-activating andstasis-eliminating also made many positive effects in antifibrosis,thecharacteristics of pharmacological effects caused by TCM are the effects onvarious levels,different stages,and many targets simultaneously,which alsohave good preventing against HF,and low toxicity to human body.a series ofresearches have indicated:biejiajian pills have showed the positive effects oninhibiting the synthesis of collagens and HSCs activation and improving liverfunction.but the active mechanism has been a difficult point and a hot point inthe research of TCM.Then the study was designed to investigate the effect ofon TGF-β1smad signaling pathway,the synthesis of collagens and theactivation and proliferation of HSCs,compare the results and mechanism withrosiglitazone.Method：Twenty-five rats were randomly divided into five groups,the preparationof biejiajian pills and rosiglitazone pharmacacentical suspension was done bymixing with sterile distilled water.the biejiajian pills were taken separately inaccordance with the adult dosage of3.5,7,14times as traditional Chinese medicinein low,middle,high dose group; rosiglitazone were taken in accordancewith the adult dosage of7times as western medicine group; The rats of controlgroup were gavaged with the same volume of distilled water. We prepared ofdrug serum after drawing blood from the inferior vena cava after seven timesdrug delivery,then10%rat medicated serum culture medium were prepared,the control group only contain normal serum of rats.the model group containnormal serum of rats, mixed with5ug/L TGF-β1. the model group and thefour treatment group need firstly add the culture medium of controlgroup,after24hours cultivating with it,then join normal serum–DMEM culture medium and drug serum–DMEM culture medium of every treatmentgroup in it respectively after discarding supernatant.HSC were cultivated in the drug serum and were detected:with the helpof cell culture technology of HSCs,when the HSCs are adherent in37℃Cell culture box,replace the former culture medium with the DMEMexcluding serum,cell synchronizing for24hours,then the drug serum–DMEM culture medium were changed into the cultivation system according tothe above mentioned method and the HSCs were observed and detected after24hours:①optical density(OD): method of MTT②COLIV,LN：radioimmunoassay③TβR I,TBRⅡ,smad3,smad7：reverse transcription-polymerase chain reaction(RT-PCR)Results:1the detections by MTT showed that: compared with the controlgroup,the OD in model group and every therapy group (except biejiajianpills in high dosage)remarkably increased(P＜0.05); compared with themodel group, the OD in every therapy group remarkably decreased(P＜0.05);compared with rosiglitazone, the OD in biejiajian pills in high dosageremarkably decreased(P＜0.05), the OD in biejiajian pills in middle dosagehad no statistical differenc(eP＞0.05), the OD in biejiajian pills in low dosageremarkably increased(P＜0.05); compared with biejiajian pills in low dosage,the OD in biejiajian pills in middle dosage and high dosage decreased(P＜0.05);There were no statistical difference of OD between biejiajian pills inhigh dosage and the control group(P＞0.05).2the detections by radioimmunoassay showed that: compared with thecontrol group,the promotion of Col IV and LN in model group and everytherapy group remarkably increased(P＜0.05); compared with the modelgroup, the promotion of Col IV and LN in every therapy group remarkablydecreased(P＜0.05); compared with rosiglitazone, the promotion of Col IVand LN in biejiajian pills in high dosage remarkably decreased(P＜0.05), thepromotion of Col IV and LN in biejiajian pills in middle dosage had nostatistical difference(P＞0.05),the promotion of Col IV and LN in biejiajian pills in low dosage remarkably increased(P＜0.05); compared with biejiajianpills in low dosage, the promotion of Col IV and LN in biejiajian pills inmiddle dosage and high dosage decreased(P＜0.05).3the results of RT-PCR analysis of all groups showed that: comparedwith the control group, the expression of TβRI mRNA,TβRⅡmRNA andsmad3mRNA in model group and every therapy group remarkably increased,the expression of smad7mRNA in every therapy group group remarkablyincreased(P＜0.05); compared with the model group, the expression of TβRImRNA,TβRⅡmRNA and smad3mRNA in every therapy group remarkablydecreased,the expression of smad7mRNA in every therapy group remarkablyincreased(P＜0.05); the expression of smad7mRNA in control group andmodel group have no statistical difference; compared with rosiglitazone, theexpression of TβRI mRNA,TβRⅡmRNA and smad3mRNA in biejiajian pillsin high dosage remarkably decreased,the expression of smad7mRNA inbiejiajian pills in high dosage remarkably increased(P＜0.05), the expressionof TβRI mRNA,TβRⅡmRNA,smad3mRNA and smad7mRNA in biejiajianpills in middle dosage had no statistical difference(P＞0.05), the expressionof TβRI mRNA,TβRⅡmRNA and smad3mRNA in low dosage remarkablyincreased, the expression of smad7mRNA in biejiajian pills in low dosageremarkably decreased(P＜0.05); compared with biejiajian pills in low dosage,the expression of TβRI mRNA,TβRⅡmRNA and smad3mRNA in biejiajianpills in middle dosage and high dosage decreased, the expression ofsmad7mRNA in biejiajian pills in biejiajian pills in middle dosage and highdosage increased(P＜0.05).Conclusion:1TGF-β1could remarkably promote the proliferation and activation ofHSCs, increased the synthesis and Secretion of ECM in HSCs, theabovementioned effects of TGF-β1may achieve by activating the signaltransduction of TGF-β Smad conductive pathway in different sites—TβR andSmads.The negative feed back effect of I-Smad--Smad7on TGF-β Smadconductive pathway can not be sustained,which may be one reason for continued development of liver fiver fibrosis.2The drug serum of rosiglitazone and biejiajian pills in different dosagescan inhibit the above effects of TGF-β1,the effects may due to inhibiting thesignal transduction of TGF-β Smad conductive pathway,but further researchis needed to prove if the effect on downstream factor is the cascade reaction ofthat on upstream factor.3The above two drugs could not make the state of HSCs resume to thelevel of the control group,or completely block TGF-β Smad conductivepathway after stimulation of TGF-β1,this may be the important reason ofdifficulty to fully respair liver hepatic fibrosis by using drugs.4The effects of biejiajian pills is dose-dependent,the effect of antihepatic fibrosis increased with the drug concentration increased gradually. |
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