The Clinical Effects And TGF-β1/Smads Signal Pathways Research Of Jia Wei Yin Chen Si Ni Decoction In Delaying Liver Fibrosis For Biliary Atresia

This thesis is supported by the funding from the national natural science foundation of China which title is "Mechanism Study of jiawei yinchen sini Decoction on TGF-β1/Smads Signal Pathways and MMPs/TIMPs in Biliary Atresia Model of Liver Fibrosis" (Project number:81373686)。Whether TGF-β1/Smads signal pathways activated or not is associated with the occurrence、development and the end outcomes of liver fibrosis, which plays a key role in regulating process of liver fibrosis due to chronic liver inflammation. In microenvironment formed by inflammation of liver fibrosis, cytokines TGF-βcan be secreted by kinds of cells such as platelets and Kupffer cells and so on in which activated Hepatic Stellate Cells secrete higher than others. Extracellular matrix main secreted by hepatic stellate cells. Therefore,we believe that TGF-βas a positive adjustment factors in the forming process of extracellular matrix. However, during 3 kinds of subtypes of TGF-β,TGF-β1 is the clear early cell factor which can caused fibrosis, the activation mechanism is TGF-β1 combined with its receptors formed complex which activate the Smads protein within the cell cytoplasm. Therefore, TGF-β1/Smads signaling pathways play an important role in forming fibrosis caused by liver inflammation, the signaling pathways also a focus in the study of liver fibrosis currently, and traditional Chinese medicine can effectively delay the occurrence and development of liver fibrosis and even can effectively reverse early liver fibrosis. Therefore, to explore the mechanism of traditional Chinese medicine have important clinical significance.Kasai procedure is the major treatment method for biliary atresia, however, which can not prevent the occurrence of liver fibrosis. Ursodeoxycholic acid and glucocorticoid are the major drugs in treating biliary atresia after Kasai procedure, but lacking long-term clinical benefit evidence in patients of biliary atresia.The treatment and research of traditional Chinese medicine for the disease started relatively late. Our research group performed a further study for syndrome of postoperative on the basis of previous studies, the results showed that the patiants with biliary atresia had characteristics of Yang deficiency、wet trapped、blood stasis, according to the characteristics of the pathogenesis, using method of wenyang huashi huoxue, and applying drugs based on yinchen sini Decoction to treat biliary atresia of postoperative, which had a certain clinical curative effect on improving clinical symptoms and the quality of life、delaying liver fibrosis, however, its mechanism is unclear, and it has not yet been reported about mechanism of treating biliary atresia with liver fibrosis.Objectivewe will perform a study about the effect of Jia Wei Yin Chen Si Ni Decoction on liver fibrosis in TGFβ1/Smads of HSC-T6 on the basis of previous research. To explore its possible mechanism and therapeutic targets, and to provide evidence of using Chinese medicine treating biliary atresia with postoperative.Methods1. Collect patients with biliary atresia from outpatient and inpatient department of first hospital affiliated Zhongshan university and the first hospital affiliated Guangzhou university of TCM, in according to the inclusion criteria and exclusion criteria included, fill in patient’s clinical symptoms intervented by Jia Wei Yin Chen Si Ni Decoction according to requirements of the clinical observation table, to evaluate efficacy of drug by statistical analysis.2. Discussing the effect of serum containing Jia Wei Yin Chen Si Ni Decoction on activation and growth in HSC-T6.2.1 The method of making serum containing drugs 60 SD rats divided into 3 groups according to random number table:blank group, high dose group, positive drug group, and drug dose with rat taking according to literature "equivalent dose conversion between animal and human body in pharmacological experiment", of which high dose group rats were given 2 times the dose of normal Chinese medicine to make high dose serum containing drug; Blank group was given the same volume distilled water as the traditional medicine; positive drug group was given the normal dose; all rats were given 2 times a day for 3 consecutive days, the fourth day’s morning give 1 time, taken abdominal aorta blood within 2 h, then centrifugal 30 min,3000 r/min, mixed serum which in the same group, inactivated 30 min within 56℃ water, removed bacteria using 0.22 um needle filter, finally saved repackaging-20℃. Dilution by blank serum before using.2.2 Method of divided groups Blank group(5ml DMEM containing 10% blank serum)、model group(5ml DMEM containing 10% blank serum)、positive drug group(5ml DMEM containing 10% positive drug serum)、high dose serum of Jia Wei Yin Chen Si Ni Decoction(5ml DMEM containing 10% high dose serum).. middle dose serum of Jia Wei Yin Chen Si Ni Decoction(5ml DMEM containing 10% middle dose serum)、low dose serum of Jia Wei Yin Chen Si Ni Decoction(5ml DMEM containing 10% low dose serum), in addition to the normal group, the rest of the groups, respectively, to join TGF-β1 (final concentration is 10 g/L), and cells of all of the groups were put in 5% CO2 incubator;2.3 Method of detecting absorbance by CCK8 Cells incubated into 96 orifice which per orifice containing 5000/ml cells, the total volume was 100 ul, every group had 5 same orifice; cultivated in incubator for 48、72 hours respectively, and added 10ul CCK8 to each orifice for half hour, detected absorbance values using 450 nm wavelength,the result described by the mean of 5 absorbance values.3. Detected expression of mRNA of TβR1, Smad3, Smad7, collagen Ⅰ, collagen Ⅲ in HSC-T6 which cultivated in serum containing drugs intervented by RT-PCR;3.1 Methods of making serum containing drugs as the same 2.1 and divided groups as the same 2.2;3.2 Cells of all of the groups were put in 5% CO2 incubator for 72 h(cells number>1×105/ml), Washed and abandoned three times with PBS, transferred to the 1.5 ml EP, centrifugal 4000 r/min 4℃ for 3 min and abandoned supernatant. Detecting mRNA expression of TβR1、Smad3、Smad7、collagen Ⅰ、 collagenⅢ by RT-PCR, and using β-action as reference, analysising by method of 2-ΔΔCt.4. Detected expression of protein of TβR1, Smad3, Smad7, collagen Ⅰ, collagen Ⅲ in HSC-T6 which cultivated in serum containing drugs intervented by Western blot.4.1 Methods of making serum containing drugs as the same 2.1 and divided groups as the same 2.2;4.2 Cells of all of the groups were put in 5% CO2 incubator for 72 h(cell number>1×105/ml), Washed and abandoned three times with PBS, transferred to the 1.5 ml EP, centrifugal 4000 r/min 4℃ for 3 min and abandoned supernatant. Detecting protein expression of TβR1、Smad3、Smad7、collagen Ⅰ、collagenⅢ by Western blot, and using β-action as reference, analysising by method of ratio of protein grayscale results.Results1. Jia wei Yin chen Si Ni Tang can effectively improve the patient’s degree of jaundice, mental state, degree of abdominal distention, vomiting, appetite, stool color character, and children liver splenomegaly and other symptoms, signs, improved the quality of life to a certain degree2. Compared with blank group, after exposure to exogenous TGF-β1 (lOng /ml) for 48 hours, each group with different dosages of medicated serum showed inhibitory effect on cell proliferation(P<0.05). However, no significant inhibitory effect were observed among them after exposure to exogenous TGF-β1(10ng/ml) for 72 hours in comparison with that of. control group.3.①There was no significant difference in the TβR1 mRNA expression between TGF-β1-induced HSC-T6 cells of model group and the blank group (P> 0.05). Compared with blank group, no significant changes were observed on the TβR1 mRNA expression of TGF-β1-induced HSC-T6 cells of the three different dosage groups of Jia wei Yin chen Si Ni Tang and the positive control group either. ②The smad3 mRNA expression in TGF-β1-induced HSC-T6 cells between the model group and the blank group is significant and the difference is also significant between the medium dosage groups of Jia wei Yin chen Si Ni Tang and model group (P<0.05);③However, there was no significant difference in the smad7 mRNA expression between TGF-β1-induced HSC-T6 cells of model group and the blank group (P> 0.05), no significant changes were observed on the smad7 mRNA expression of TGF-β1-induced HSC-T6 cells of the three different dosage groups of Jia wei Yin chen Si Ni Tang and model group either.; ④There was significant difference in the collagen I mRNA expression between TGF-β1-induced HSC-T6 cells of model group and the blank group and between the medium dosage groups of Jia wei Yin chen Si Ni Tang and model group (P <0.05);⑤The collagenⅢ mRNA expression in TGF-β1-induced HSC-T6 cells between the model group and the blank group is significant and the difference is also significant between the high dosage group of Jia wei Yin chen Si Ni Tang and the model group (P<0.05)4.①The results of Western blot for TβR1 protein expression:multiple comparison of the relative expression of TβR1 protein between blank group and model group and between low dosage group and model group is significant (P<0.05);②The results of Western blot for Smad3 protein expression: multiple comparison of the relative expression of Smad3 between blank group and model group and between low dosage group and model group is significant (P<0.05);③The results of Western blot for Smad7 protein expression: There was no significant difference in the Smad7 expression between each groups (P>0.05);④The results of Western blot for collagen Ⅰ protein expression: there was no significant difference in the collagen Ⅰ expression between each groups (P>0.05);⑤The results of Western blot for collagenⅢ protein expression:compared with model group, there was significant in the relative expression of collagenⅢ protein in the blank group, positive group and medium dosage groupt (P<0.05).ConclusionIn summary, Jia wei Yin chen Si Ni Tang treatment postponed the occurrence and progression of liver fibrosis and improve the outcomes through the mechanism involving TGF-β1/Smads Signaling Pathway. We found that smad3 mRNA、 collagen Ⅰ mRNA、collagenⅢ mRNA expression of Jia wei Yin chen Si Ni Tan-treated groups were significantly lower than those of controls (P<0.05). In addition, as compared to controls, TβR1、Smad3、collagenⅢ protein expression also decreased (P< 0.05). It was, therefore, concluded that Jia wei Yin chen Si Ni Tang treatment inhibited the progression of liver fibrosis and improve the outcomes through the mechanism involving down-regulation of TGFβ1/Smads and decreasing the expression of collagen. However, no significant changes were observed in terms of the Smad7 mRNA/protein expression which is inconsistent with our expected results. This could be that the existence of multiple targets in the interactions between the body and Jia wei Yin chen Si Ni Tang treatment which may provide new insight into the further investigations.