| Objective Liver fibrosis is the process of over-precipitation of extraculluar matrix (ECM) in liver tissue, which is induced by series of damage-recovery caused by different chronic liver disease. The mechanism of development of liver fibrosis is quite complicated and remains mostly unknown for this process which limits the development of therapy. Hepatic Stellate cell, HSC, a main component of ECM, is considered as the center related to the development of liver fibrosis caused by liver damage. Extracellular Matrix plays an important role for the initiation and maintainance of HSC during the development of liver fibrosis. The complex relationship between HSC and ECM is mainly induced by integrin. Focal adhesion kinase, FAK is a mediator of integrin signaling pathway which connects integrin and its downstream molecules, and a cross point of other different signaling pathways. Among them, Ras-MAPK is mainly pathway involved in integrin signaling, and extracellular signal-regulated kinase, ERK1/2 is one of well-studied member of MAPK family. Ras-RAF-MEK-ERK pathway is related to many activities in cells. KangXian Ruangan Granule (KXR), which has good clinic therapy effect, has been prepared as granule. In the present research, based on the interaction between HSC and EcM, KXR effect on integrin signaling including integrin, FAK, ERK1/2 in Hepatic stellate cell was studied using in vitro cell-culture technique. The mechanism of KXR action was proposed to relate to anti-proliferation of HSC and induction of apotosis.Methods (1) The technologyof preparation of KXR was optimized based on the chemical, physical character and pharmacological action of radix salviae miltiorrhizae, hawthorn and carapax trionycis. TanshinoneⅡA and rhizoma curcumae oil were extracted and the amount of several supplement material was optimized based on The technological process of dispersible tablet. Preparation recipe was finally designed.(2) The dissolution in vitro of finished product was investigated. The quality was controlled and the standard of quality was established.(3) Different drugs(FN+10%NRS,FN+5%kxr,FN+10%kxr,FN+20%kxr) was added on HSC-T6 cell line and compared to that of control. The FN (3μg/ml) was pre-treated 30min before kxr was added, cells were cultured another 48h.(4) The proliferation of cell was estimated using MTT method; And the adhesion rates of cell were observed by toluidine blue colorimetric assay; Hsc apotosis was measured using V-PE/7-ADD method.(5) The expression of integrin alpha5 beta1 protein was measured using immunochemistry method.(6) The expression of FAK protein was measured using immunochemistry method, the mRNA of FAK was investigated using RT-PCR method(7) The expression of ERK1 protein was measured using immunochemistry method, the mRNA of ERK1 was investigated using RT-PCR methodResults (1) The consistency of alcohol was 90%, the flow was 0.6mi/min, the amount was 12 times, the immersion was 16h; Carapax Trionycis was vltram icro-porphyrized and dectected under the microscope, the results show that 50% of powder were smaller than 5um, and 95% of them were smaller than 10um; the optimum extraction condition of Rhizoma curcumae oil was 8 times water, steeping 0.5 hours, boiling 5 hours. In the preparation of the inclusion, the optimum inclusion condition was as follows; the ratio of oil andβ-CD, 1 to 4; inclusion temperature 55℃; stirring time 60min. In the preparation of the granula, dextrin was used as adjuvant, 0.5 times of the powder amount; 50% alcohol was used as wetting agent; CMC was added in as suspenging agent, 0.4% times of the powder amount.(2) The indexes of the product didn't obviously change in room temperature and the surrounding with 40℃and RH(75%) last 6 months.(3) Comparing to FN treatment, Kxr treatments suppressed the proliferation of HSC, and induced the apotosis of HSC with statistical significance (P＜0.05). All Kxr treatments suppressed the attachment of FN and HSC significantly (P＜0.05) comparing to that of control group.(4) The protein level of integrin alpha5 betal of HSC from KXR-treated serum decreased significantly comparing to control (p＜0.05) and FN group (p＜0.01). (5) The mRNA and protein level of FAK of HSC from KXR-treated serum dropped significantly comparing to control, KXR1 (p＜0.05) and KXR2,KXR3 (p＜0.01) group, and decreased 43.32%(11.93%), 56.52%(39.52%), 70.27%(53.42%) respectively.(6) The mRNA and protein level of ERK1 of HSC from KXR-treated serum dropped significantly comparing to control, KXR1 (p＜0.05) and KXR2,KXR3 (p＜0.01) group, and decreased 39.53% (8.93%), 47.45% (35.62%), 56.78% (50.12%), respectively,Conclusion (1) The optimized technologyis stable and reliable, and the quality control is reasonable.(2) FN stimulated the proliferation of HSC which indicates that extracellular matrix plays important roles in initiation and maintainance of HSC induced by integrin. We found KXR-treated serum suppressed the proliferation of HSC stimulated by FN, induced the apotosis of HSC and suppressed the attachment of HSC in dose-dependent manner. KXR decreased the protein level of integrin alpha5 beta1 of HSC and suppressed the expression of FAK and ERK1 at both transcription and translation level.(3) KXR-treated serum has good effect on proliferation of HSC and induction of HSC apotosis. KXR played its action probably by the following pathway. It suppresses or limits the combination between EM and its receptor integrin and further suppresses the adherence of EM and HSC. By decreasing protein level of integrin, KXR reduces both mRNA and protein expression level of FAK and ERK1 and further suppressed integrin related Ras-dependent MAPK pathway in FAK signaling, that is Ras-RAF-MEK-ERK pathway which is possible target point of anti-liver fibrosis action.
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