Correlation Between Hepatic Stellate Cells In The Wnt/β-catenin And Wnt/Ca²⁺ Signaling Pathway Between The Intervention Under SB216763

Objective: Liver fibrosis is a chronic liver disease eventually progress to livercirrhosis, an important intermediate process in hepatic stellate cells (hepatic stellate cell,HSC) of activation and extracellular matrix (extracel lular matrix, ECM) the largeamount of deposit is a prominent feature. Studies confirm that Wnt signaling pathwayplays an important part in the process of liver fibrosis, the Wnt/beta-catenin study most.Chinese medicine liver convalescent is pathophysiological teaching and research sectionof our school after years of anti liver fibrosis was concluded the experience of the party,has confirmed the Wnt/beta-catenin pathway and the classical Wnt/Ca2+signalingpathways have inhibition. Currently, found in the study of some tumors,Wnt/Ca2+signaling pathways of Wnt/beta catenin pathway can have the effect ofinhibition, which illustrates the two pathways in some tumor is not a single effect, and isalso there may be some relationship between two pathways, able to influence each other,but in the study of liver fibrosis is the main research of each pathway and mechanism ofthe occurrence of liver fibrosis development there may be some, but for the relationshipbetween the two pathways are still not very clear. This experiment by using Chinesemedicine liver convalescent medicine serum and HSC SB216763intervention training,then analysis in the process of liver fibrosis development Wnt/beta-catenin pathwayand Wnt/relationship between Ca2+signaling pathways. Methods: Rat HSC-T6cell lines with DMEM medium containing10%fetalbovine serum in37℃and5%CO2gas mixture culture incubator, batches. Treatedcells when conditions are ripe, reoccupy DMEM medium without serum starvationgroup after24h dosing.The cells in six orifice were randomly divided into the following five groups:normal serum group (DMEM medium containing10%normal rat serum); SB216763(including20M group (containing10%normal rat serum, DMEM medium ofSB2167632umol/L); SB216763(including10M group (containing10%normal ratserum, DMEM medium of SB2167631umol/L): GanFuKang treatment group(containing10%liver convalescent treatment group rats serum DMEM medium);SB21676320including GanFuKang treatment group (20(including SB216763with10%GanFuKang drug intervention on the basis of M) in37℃and5%CO2gas mixtureof incubator in48h.Reverse transcription-polymerase chain reaction (reverse transcriptase polymerasechain reaction, RT-PCR) method for determination of collagen in HSC cell I, collagenⅢ, the expression of a-SMA and Wnt/Wnt5a Ca2+signaling pathways relevantindicators, Frizzled2, CAMKII, mRNA expression of MPP-7, immune proteinimprinting method (western blot) protein expression of MPP-7observation cells. Theexperimental results using SPSS13.0statistical software analysis.Results:1．SB216763and GanFuKang of rat HSC T6cell activity and a-SMA, theinfluence of the type I and type III collagen1.1SB216763RT-PCR detection of rat HSC T6cell activity and a-SMA, theinfluence of the type I and type III collagenSB216763(including10M and HSC SB216763(including20M group wasobviously activation, type I collagen, collagen type Ⅲ, a-SMA expression quantitycompared with normal serum group increased significantly, which in SB216763(including20M, and the more obvious the increase of group use SB216763HSC-afterT6cell lines have obvious tendency of fibrosis. 1.2RT-PCR detection of GanFuKang in HSC-T6type I collagen, collagen typeⅢ, a-SMA expressionLiver convalescent serum drug group and SB216763(including10M, comparedSB216763(including20M group, HSC cells of type I collagen, collagen type Ⅲ, theexpression of a-SMA amount is significantly reduced, at the same time SB216763(including20M plus liver convalescent serum group compared with SB216763(including20M group, the expression of quantity are reduced, and illustrate liverconvalescent can effectively inhibit the type I collagen, collagen type Ⅲ, a-SMAexpression in HSC-T6cell lines.2．SB216763and GanFuKang of HSC-T6Wnt/Ca2+signaling pathways in celllines express the influence of related indicators2.1RT-PCR detection Wnt/Ca2+Wnt5a mRNA expression in the signal pathWnt5a is Wnt/Ca2+signaling pathway is an important initial protein, in normal ratliver cells, and so almost no expression. Although Wnt and different receptors, canactivate different Wnt pathways, including classic Wnt pathways, but in this experiment,the classic Wnt pathways activated, by rt-pcr showed that each group of Wnt5a mRNArelative expression has no obvious difference compared with control group, there is nostatistical significance.2.2RT-PCR detection Wnt/Ca2+Frizzed2mRNA expression in the signal pathFrizzed2is Wnt signaling pathways of a kind of transmembrane receptor protein,combination Wnt5a and activation of Wnt/Ca2+signaling pathways, expressed in normalrat liver cells are very few, almost no expression. Pathways rt-pcr test its SB21676320including M, including10M, liver convalescent medicine treatment group, liverconvalescent serum plus SB216763mRNA relative expression compared with normalserum group has no obvious change.2.3RT-PCR detection Wnt/Ca2+CAMKII mRNA expression in the signal pathCAMKII is key to Wnt/Ca2+signaling pathways downstream of a protein kinase,expressed in normal rat liver cells trace, almost no expression. Through rt-pcr detectionCAMKII relative expression of mRNA in each group, found no expression differences compared with normal serum group, no statistical difference, that after the use ofSB216763, Wnt/Ca2+signaling pathways may not be affected by Wnt/beta Cateninsignaling pathways and the corresponding change.2.4RT-PCR detection of Wnt/Ca2+signaling pathways mRNA expression ofMMP-7MMP-7expressed in normal rat liver cells are very few, almost no expression. Inrt-pcr detection results, although compared with normal serum group,20(includingSB216763mRAN express relative increase in M group, but there is no significantdifference. SB21676310in other three groups (including M, liver convalescent serumdrug group and convalescent SB216763(including20M mRNA in the relativeexpression quantity had no obvious change.3．Western blot test resultsMPP-7rarely expressed in normal liver tissue cells, almost no expression.Compared with normal serum group of SB216763(including20M more evident,including10M and within the liver, liver convalescent, no obvious difference, plusSB216763expression and expression are not obvious, consistent with the results ofrt-pcr, this might be related to its complex regulation.Conclusion:1. the classic Wnt signaling pathways activated, can increase the rat HSC-T6cellactivity; GanFuKang on activation of rat HSC-T6cell has certain inhibitory effect.2. classic Wnt signaling pathways activated after the classic Wnt/Ca2+signalingpathways may not occur at the same time corresponding activation or inhibition ofchange.