The Study Of The F Unction And Mechanism Of Compound Phyllanthus Urinaria (CPU â…¡)Inhibit HSC-T6 Proliferation And Induce Apoptosis

ObjectiveThe foundation of compound Phyllanthus Urinaria Ⅱ(CPUⅡ) was based on the theory of TCM and the experience of treating chronic hepatitis and liver fibrosis by screening and optimizing its components for many years. CUPⅡ was composed of ye xia zhu (Phyllanthus Urinaria), ku shen(Sophora flavescens Ait.L), tian san qi(radix notoginseng), tao ren(semen persecae), tai zi shen(Pseudostellaria heterophyla Pax ex Paxet Hoffm),cai hu(Bupleurum chinense DC) and so on. This compound recipe have been demonstrated that have the better effectiveness of anti-experimental liver injury and liver fibr-osis.Through the in vitro studies to observe the influenceof the bead compound leaves Ⅱ to HSC-T6 cell proliferation and apoptosis and the changes of expression of TGF-β1 collagen I-miR-16 and BCL-2. It aimed to provide proof of treating liver fibrosis with CUPⅡ in clinic trail.Exper imental contents1. Compound of Phyllanthus Urinaria II,in Hepatic stellate cells growth inhibition and induction of apoptosis in vitro. 1.1 Study of the effect of Compound of Phyllanthus Urinaria Ⅱ on proliferation inhibition.Hepatic stellate cells were used as model. By testing the OD value of different groups at the time of 72h(corresponding TC50 3.72mg/ml). HSC-T6 cells were cultured in vitro and divided into 4 groups, including control group, CPUⅡ 3.60 mg/ml group, CPU Ⅱ 1.8mg/ml group, CPU Ⅱ 0.90mg/ml group, and MTT colormetric assay was used for evaluating the inhibitive effect on HSC-T6. we found that there was a significant time-dose relationship between Compound of Phyllanthus Urinaria Ⅱ and the proliferation inhibition(P<0.05).1.2 Study of the effect of Compound of Phyllanthus Urinaria Ⅱ on the cycle.Determining the HSC-T6 cell which was intervened 72h by Compound of Phyllanthus Urinaria Ⅱ through flow cytometry assay Annexin V-FITC/PI. The results show that cells percentage of the dosage group and high dose group was obviously increassing in the G0/G1 phase, comparing with control group(P<0.01). Cells percentage of the dosage group and high dose group was obviously reducing in the S phase, comparing with control group(P<0.01). Cells percentage of the dosage group and high dose group was obviously decreassing in the G2+M phase, comparing with control group(P<0.01).1.3 Study of the effect of Compound of Phyllanthus Urinaria Ⅱ on apoptosis induction.Determining the HSC-T6 cell which was intervened 72h by Compound of Phyllanthus Urinaria Ⅱ through flow cytometry assay Annexin V-FITC/PI. Apoptosis rate was higher with the increasing concentration of Compound of Phyllanthus Urinaria Ⅱ (0.9mg/ml、1.8mg/ml、3.6mg/ml), which showed a statistic difference (P<0.01), and there was also a time-dose relationship.1.4 Study of the influence of Compound of Phyllanthus Urinaria Ⅱ on the expression of TGF-β1mRNA and collagen ImRNA.HSC-T6 cells were cultured in vitro and divided into 4 groups, including control group, CPU Ⅱ 3.60 mg/ml group, CPU Ⅱ 1.8mg/ml group, CPU Ⅱ 0.90mg/ml group. The level of TGF-β1mRNA and collagen ImRNA were analyzed by real time PCR; The results showed that CPUII can decreases the expression levels of TGF-β1mRNA and collagen ImRNA comparing with those in the cintrol group (P<0.05,0.01). The expression level of TGF-β1mRNA and collagen ImRNA was decreasing with the increasing concentration of Compound of Phyllanthus Urinaria Ⅱ(0.9mg/ml、1.8mg/ml、3.6mg/ml), which showed a statistic difference (P<0.01), and there was also a time-dose relationship. It suggest that CPUII possess the anti-liver fibrosis by decreasing the expression level of TGF-β1mRNA and collagen ImRNA.1.5 Study of the influence of Compound of Phyllanthus Urinaria Ⅱ on the expression of the proteins of collagen l.HSC-T6 cells were cultured in vitro and divided into 4 groups, including control group, CPU Ⅱ 3.60 mg/ml group, CPUⅡ 1.8mg/ml group, CPU Ⅱ 0.90mg/ml group. The level of collagen I proteins were analyzed by Western blotting. The results showed that high, middle and the low dosage groups of CPUⅡ all have remarkable inhibitive effect on the proteins of collagen I comparing the control group(P<0.01).2. Impact of compound phyllanthus urinaria (CPU Ⅱ) on the expresstion of TGF-β1 and collagen Ⅰ synthesis in hepatic stellate cells.2.1 Study of the influence of Compound of Phyllanthus Urinaria Ⅱ on the expression of miR-16mRNA and BCL-2mRNA.HSC-T6 cells were cultured in vitro and divided into 4 groups, including control group, CPUⅡ 3.60 mg/ml group, CPUⅡ 1.8mg/ml group, CPUⅡ 0.90mg/ml group. The level of miR-16mRNA and BCL-2mRNA were analyzed by real time PCR;The results showed that CPUII can increases the expression levels of miR-16mRNA and decreases the expression levels of BCL-2mRNA comraring with those in the control group(P<0.05,0.01).2.2 Study of the influence of Compound of Phyllanthus Urinaria Ⅱ on the expression of the proteins of BCL-2.HSC-T6 cells were cultured in vitro and divided into 4 groups, including control group, CPUⅡ 3.60 mg/ml group, CPUⅡ 1.8mg/ml group, CPUⅡ 0.90mg/ml group. The level of BC1-2 proteins were analyzed by Western blotting.The results showed that high, middle and the low dosage groups of CPUⅡ all have remarkable inhibitive effect on the proteins of BCL-2 comparing the control group(P<0.01).Conelus i onCPUII have the well capability of antifibrosis. The inhibitory effect of Compound of Phyllanthus Urinaria Ⅱ on HSC-T6 cells achieved by inducing apoptosis; CPUⅡ is capable of regulating the expression of TGF-β1and collagen I synthesis in hepatic stellate cells and have good regulation on miR-16 and BCL-2, this two-way adjustment play an inhibitory roleimportant role on increasing antifibrosis.