Interruptting Effect Of Qing Gan Hua Yu Solution On Rat Hepatic Precancerous Lesion And Regulatory Effect On Notch Signaling Transduction Pathway

Hepatocellular carcinoma (HCC) is one of the highest morbility malignant tumors in the world, and about 100 thousands new cases occur in china every year., The mortality rate of HCC has become the second place of all malignant tumors in our country. Operation excision is the first way to cure this disease,but the relapse rate is high, and long-term effect is not definite, whose treatment is still in trouble. Resently, with the establishment of synthetical therapy concept , the traditional Chinese medicine has been taken to prevent and treat HCC actively. Combined with operation,radiotherapy and chemotherapy, therapeutic effect of HCC has been improved. Qing Gan Hua Yu Solution was created by Professor Yao Shukun according to the theory of Chinese traditional medicine and clinical study over many years ; The therapeutic principles are deintoxicating and damping elimination , activating blood circulation to dissipate blood stasis, invigorating the spleen and regulating qi. Studies have proved that Qing Gan Hua Yu solution have obvious interruptting effect on HCC and hepatic precancerous lesion .Qing Gan Hua Yu Solution can inhibit the formation and progress of HCC through mult–links,mult-targets.At present, the developing mode of tumer is a mult-genic mutations,mult- factors,mult- pathways, mult- procedures process. In this process, some important growth factor and receptor regulate cell proliferation, differentiation, development, apoptosis through signaling transduction pathway.Abnormal cell signaling transduction will induce tumer to proliferate rapidly and grow unlimitedly. With the development of molecule oncology, molecule pharmacology , the function of signaling transduction in tumer formation has elucidated, the signaling transduction has become new target in medicine research about tumer treatment. Resent years studies show that Notch signaling transduction pathway play an important role in several kinds of tumour's formation and how does it influence on HCC and hepatic precancerous lesion ? There is not so many relative reports.Recent years, the special pathological change of hepatic precancerous lesion Has drawn worldwise experts a great attention . Scholars discovered that cell appearance has not changed, but some abnormal characters about enzymology and protein has occurred. These cells will become carcinoma cells under some factors. To research deeply pathological mechanism of this phase will provide theoretical proof in order to intervene and reverse this lesion and interrupt the formation of HCC. Therefore, we investigate some primary pathological changes and the alteration of Notch signaling transduction pathway about hepatic precancerous lesion by enzyme-histochemistry and immunohistochemistry, immunocytochemistry, reverse transcriptase-polymerase chain reaction ,observe the interruptting effect of Qing Gan Hua Yu Solution on hepatic precancerous lesion and the regulatory funtion on Notch signaling transduction pathway. Further more, serum pharmacological method has been used to explore the function of Qing Gan Hua Yu Solution inducing hepatic oval cell line WB-F344 cell differentiation and becoming mature hepatic cell, which indicated whose function was related to regulating Notch signaling transduction pathway.Part One Interruptting effect of Qing Gan Hua Yu Solution on rat hepatic precancerous lesionObjective To observe the changes of morphology, cell ultrastructure, enzymology, protein expression of rat hepatic precancerous lesion. To investigate some primary pathological change of rat hepatic precancerous lesion and interruptting effect of Qing Gan Hua Yu Solution on rat hepatic precancerous lesion.Methods1. 90 rats were divided into four groups: A: model group; B: Qing Gan Hua Yu Solution equal effective dosage group; C: Qing Gan Hua Yu Solution high dose group D:matrine group E: normal control group . Hepatic precancerous lesion models of group A,B,C,D of the rats were induced through injecting diethylnitrosamine ( DEN) into abdominal cavity and excising 2/3 liver; At the same time , the rats of group B,C,D were intragastric administrated with Qing Gan Hua Yu Solution by different doses and matrine. At the end of seven weeks the experiment was terminated.2. Liver tissue morphological changes of rats in different groups were observed through HE stain; Cell ultrastructure was observed under transmission electron microscope ; Distribution and location of glutamyl-transpeptidase (γ-GTase), adenosine triphosphatase (ATPase), glcose-6-phosphatase (G-6-Pase) were detected through enzyme- histochemistry method; Distribution and location of Thy-1, ALB, AFP protein were detected by immunohistochemistry method.Results1. Hematoxin eosin (HE) stain:at the end of seven weeks the hepatic lobules structure of group A disappeard,the small bile duct proliferated in portal area , considerable oval cells proliferated beside small bile duct,some oval cells proliferated and inserted liver parenchyma and destroied adjacent hepatic plate; The hepatic lobules structure in group B,C,D existed, there was small amount variation hepatocytes and small amount of proliferated oval cells, the function of group C was conspicuous.2. Transmission electron microscope: oval cells were divided into three types: the cell cubic capacity became bigger and bigger form typeⅠto typeⅢ, the structure of organelle became more and more abundant, the tonofilaments and desmosome juntion were distinctive structure of oval cell.3. Enzyme- histochemistry stain:γ-GTase :There were many ellipse positive reaction focus surrounding portal area or in hepatic lobules, the focus of disease focus in groupB, C, D was dimmer and smaller. The disease focus area of unit area (mm2/cm2 ) of group B, C, D were: 8.24±0.53, 4.39±0.48, 3.16±0.53, 3.25±0.47; GroupB,C,D had evident funtion of inhibiting onγ-GTase disease focus,(P <0.05 or P <0.01),the funtion of group C,D was more obvious than that of group B, there was not evident difference between group C and group D (P >0.05). ATPase,G-6-Pase: The ellipse negative disease focus in hepatic lobules had been observed. The disease focus area of groupB,C,D had been diminished.4. Immunohistochemistry stain : The Thy-1, AFP protein expression of group E was negative, the Thy-1,AFP protein expression of group A in portal area and hepatic lobules was high, which of group B,C,D were down-regulated . The ALB protein expression of group E was positive, which of group A was down-regulated , which of group B,C,D were up-regulated, the optical density value of group A,B,C,D were: Thy-1: 150.49±9.45, 135.16±6.15, 103.83±3.72, 102.70±7.75, 79.19±6.32 ;AFP :130.34±10.50 , 113.38±8.20 , 89.29±7.05, 84.94±5.08, 62.82±7.00 ;ALB :108.79±9.31, 130.50±12.36, 157.47±6.81,159.86±9.74, 186.84±9.75. There is significant difference between group B,C,D and group A, the funtion of group C,D was more obvious than that of group B(P <0.01), there was not evident difference between group C and D(P >0.05).Conclusions1. Rat hepatic precancerous lesion appeared typical primary pathological changes, oval cell proliferation was the distinctive chagement . Qing Gan Hua Yu Solution can mitigate these pathological changes.2. According to the results of enzyme-histochemistry stain, we presumed that mechanisms of hepatic precancerous lesion were over-transport and synthesis of amino acid molecule, Ca2+ inflow, glycometabolism disturbance, Qing Gan Hua Yu Solution can possibly interrupt precancerous lesion through inhibiting over-transport and synthesis of amino acid molecule, inhibiting Ca2+ inflow , making glycometabolism stable .3. According to immunohistochemistry stain results ,we presumed that there were considerable primal stem cells in hepatic precancerous lesion phase , which expressed some characters of hepatic carcinoma cells, Qing Gan Hua Yu Solution and matrine can possibly induce hepatic stem cells into mature hepatic cells.Part Two The effect of Qing Gan Hua Yu Solution on Notch signaling transduction pathway of hepatic precancerous lesion ratsObjective To investigate the expressions of Notch signaling molecule protein and gene in hepatic precancerous lesion phase and regulatory effect of Qing Gan Hua Yu Solution on Notch signaling transduction, to explore the funtion of Notch signaling transduction on hepatic precancerous lesion and molecule mechanism of Qing Gan Hua Yu Solution to interrupte hepatic precancerous lesion.Methods1. The experiment groups were identical to which of part one, the location and quantitation expression of the Notch1 receptor and Jagged1 ligand in different experiment groups were observed by immunohistochemistry stain.2. Semi-quantitation expression of AFPmRNA, ALBmRNA, Notch1mRNA, Jagged1mRNA, RBP-JκmRNA, Hes-1mRNA in different experiment groups were observed by RT-PCR.Results1. Notch1 protein expressed even and a little in cell membrane and cytoplasma of liver tissue in group E, the strong positive expression in group A was observed besides portal area and in hepatic lobules; Jagged1 protein expressed in membrane and cytoplasma of endothelial cell in hepatic sinusoid, the dilute brow-yellow stain in hepatic lobules proliferation focus in group A. The area and degree of positive focus were decreased with the intervention of Qing Gan Hua Yu Solution and matrine . The OD value of group A, B, C,D were: Notch1 :181.15±8.39, 167.55±6.34 , 31.44±10.08, 124.33±6.55, 83.17±8.74 ;ALB:150.33±9.35, 135.29±9.89, 109.27±11.65, 105.16±8.88, 75.58±8.73.There was a significant difference between group B,C,D and group A(P <0.05 or P <0.01).There was not evident difference between group C and group D (P >0.05).2. The expression of AFP mRNA, Notch1mRNA, Jagged1mRNA, RBP-Jκ mRNA, Hes1mRNA in group A were high; the expression of ALB mRNA was low; Those expressions in group E were reverse; through therapy of Qing Gan Hua Yu Solution and matrine the expressions of AFP mRNA , Notch1mRNA , JaggedmRNA , RBP-JκmRNA , Hes1mRNA in group B, C, D were low than that in group A(P<0.05 or P<0.01), the expression of ALB mRNA in group B,C,D was high than that in group A (P<0.01); There is not significant difference between group C and groupD(P>0.05).ConclusionsIn hepatic precancerous lesion phase, the expressions of Notch signaling molecule protein and gene were high ,however, after therapy of Qing Gan Hua Yu Solution, the expression was low.Then we presumed that Qing Gan Hua Yu Solution can possibly interrupt hepatic precancerous lesion through regulating Notch signalling pathway.Part There Interruptting effect of Qing Gan Hua Yu Solution drug serum on proliferation of hepatic oval cell line WB-F344 cell and regulatory effect on Notch signaling transduction pathway.Objective To investigate the expressions of Notch signaling molecule on the hepatic oval cell line WB-F344 cell cultured in vitro and to explore the influence of Qing Gan Hua Yu Solution drug serum on differentiation and development of WB-F344 cell line.Methods1. Preparation and groups of medicated serum : the method of model establishment and the drugs'administration was the same as which of Part One, the experimental rats were anesthetized after intragastric administration at the end of seven weeks, blood in inferior vena cava was collected and segregated and filtrated bacterium, frozen at -80℃. Group A: hepatic precancerous lesion rats serum; group B: Qing Gan Hua Yu Solution drug serum; group C: matrine drug serum; group D: normal control rat serum2. The effect of the Qing Gan Hua Yu Solution drug serum and matrine drug serum on proliferation in WB F-344 cell line by MTT 3. The effect of the Qing Gan Hua Yu Solution drug serum and matrine drug serum on expressions of AFP,ALB,Notch1 protein on WB F-344 cell line by immunocytochemistry stain method.4. To explore the effect of the Qing Gan Hua Yu Solution drug serum and matrine drug serum on the expressions of AFPmRNA, ALBmRNA, Notch1mRNA, Hes1mRNA on WB F-344 cell line by RT-PCR.Results1. The drug serum of group A can promote WB F-344 cells to proliferate, the 5%, 10%, 20% drug serum of group B,C can inhibit proliferation of WB F-344 cells in 24h,48h,72h.In group B and C under the identical concentration,with the lasting of time ,the inhibition ratios were increased ; at the identical time, with the increasing of concentration, the inhibition ratio were increased,which indicated that the inhibitory funtion of two types of drug serums on proliferation of WB F-344 cells were depended on time and concentration.2. The WB F-344 cells cultured by the drug serum of group D expressed AFP, Notch1 protein, the expression of ALB protein was negative; the expression of AFP,Notch1 protein were increased by group A drug serum and the expression of ALB protein was still negative; In group B, C treated by the medicated serum, the expression of AFP,Notch1 protein were decreased and the expression of ALB protein appeared.The OD value of group A-D: AFP:113.17±14.25, 54.50±8.97, 73.06±6.26,35.95±4.83;Notch1:84.97±6.41 , 45.81±4.77, 61.50±50.43, 33.20±3.60; the OD value of group A was lower than that of group B, C, D(P <0.01);There was significant difference between group C and groupD(P <0.01). The OD value of ALB protein were:group B: 46.45±6.92,group C: 38.64±4.33; There was significant difference between group B and group C(P <0.01)3. The expressions of AFPmRNA,Notch1mRNA,Hes1mRNA of WB F-344 cells cultured by the drug serum of group D were slight. The expression of ALBmRNA was negative; the expressions of AFPmRNA, Notch1mRNA, Hes1mRNA of WB F-344 cells cultured by the drug serum of group A were increased, The expression of ALBmRNA was still negative; the expressions of AFPmRNA, Notch1mRNA, Hes1mRNA in group B, C were slighter than those in group D, there was significant difference between group B and group C(P<0.01)Conclusions1. The drug serum of Qing Gan Hua Yu Solution and matrine can inhibit the proliferation of WB F-344 cells, the function of Qing Gan Hua Yu Solution was stronger than that of matrine.2. The drug serum of Qing Gan Hua Yu Solution and matrine can down-regulate the protein and gene expression of Notch signaling molecules and ALB, the function of Qing Gan Hua Yu Solution was stronger than that of matrine,which indicated the drug serum of Qing Gan Hua Yu Solution and matrine can induce WB F-344 cells into mature hepatocyte, whose function was related to down-regulating Notch signaling molecules.