| Biliary atresia is one of the common causes of obstructive jaundice in neonates and infants.It refers to the stenosis or interruption of intrahepatic and extrahepatic bile ducts,which leads to the exclusion of bile and it is characterized by progressive inflammation and fibrosis.The process of liver fibrosis runs through the whole process of BA.Although it can prolong the life of some children with BA,Kasai surgery cannot reverse liver fibrosis and delay the progression of liver cirrhosis.Drugs for the treatment of hepatic fibrosis after BA postoperative were very limited.Liver fibrosis runs through the whole process of BA.In the early stage of BA,although hepatic fibrosis has not yet appeared,its related factors have been activated.Hepatic stellate cells are major players.The TGF-?1/smad pathway is one of the key factor of hepatic fibrosis and is closely related to the activation of HSCs.The deposition and degradation of extracellular matrix determine the development direction of liver fibrosis.MMP9/TIMPs is closely related to the extracellular matrix.MMP9 is a major degradation enzyme of collagen,while TIMPs is the main endogenous inhibitor of MMP9,which binds to the active site of MMPs to inhibit its activity.Because liver fibrosis is reversible to some extent,the intervention of anti-fibrotic drugs in the early stage of BA may effectively prevent and treat liver fibrosis of BA.Our team takes the lead in carrying out a series of clinical and experimental studies on the treatment of BA with traditional Chinese medicine in China.The clinical findings showed that children with BA after Kasai had the characteristics of Yang deficiency,wet sleepiness and blood stasis,and the method of warming Yang to moistening and activating blood was often used in the treatment.We used jiawei yinchensini decoction to treat BA children and found that it could improve lamps in the abdomen,abdominal distension and appetite and other symptoms and signs and liver function index,reduce the recurrence of cholangitis,effectively reduce the level of hyaluronic acid,mucin layer,collagen type ? and collagen type ? in the serum to improve liver fibrosis.At the same time,our team carried out a series of cell and animal experiments to explore the target of jiawei yinchensini decoction in the treatment of BA liver fibrosis.And we found that jiawei yinchensini decoction could reduce the expression of samd3 and collagen type ? in HSCs-T6 of rat intervented by TGF-?1 to delay the liver fibrosis in vitro,and decrease the expression of smad3,increase the expression of smad7,inhibit the expression of collagen ? and collagen ?,increase MMP1 and inhibit TIMP1 and TIMP2,to play an inhibitory effect on liver fibrosis.On the basis of previous studies,this clinical study carried out the summary of clinical symptoms of children with BA after Kasai surgery,as well as the analysis of prescription and medication law,expecting to form a systematic standard.This experimental study 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-?1/smads Signal Pathways and MMPs/TIMPs in Biliary Atresia Model of Liver Fibrosis"(Project number:81373686).Our research group has completed part of the experiments on animal models of liver fibrosis.Now we successfully established the BA neonatal mice model by intraperitoneal injection of rhesus monkey rotavirus(RRV),and explored the therapeutic target of jiawei yinchensini decoction for treating BA from the TGF-1/smad pathway and MMP9/TIMPs balance.Objective1.On the basis of previous research,"heritage of traditional Chinese medicine(V2.5)auxiliary system" software was used to analyze the symptoms,complications and Chinese traditional medicine prescription rule of children after operative of biliary atresia by data mining,to summarize and analyze the experience of lingnan Li Shi pediatric in the therapy of biliary atresia,and to provide some basis for traditional Chinese medicine in the treatment of symptoms after operative of biliary atresia.2.To observe the intervention effect of jiawei yinchensini decoction on the biliary atresia mouse model induced by rhesus rotavirus,to study its effects on symptoms,liver mass,liver index,and liver histopathology,and to explore its target from the TGF-1/smads pathway and MMP9/TIMPs.Methods1.clinical researchThis study was conducted to summarize the clinical symptoms of children with BA after Kasai and to analyze the prescription and medication rules.This study collected 33 pediatric patients diagnosed with biliary after Kasai from December 2017 to December 2018 in the first affiliated hospital of Guangzhou University of Chinese medicine.We input the cases of children with BA through"clinical acquisition module" in "Chinese auxiliary system"(V2.5)software,and analyzed the ratio of sex,symptoms,complications,frequency of drugs,four properties and five tastes and channel tropism through the"statistics of cases" module in the system of "statistical",and analyzed the prescription law and the new prescription combination through the "prescription analysis"function of "data analysis".2.experimental study2.1 BA neonatal mice mode constructed with different titers of RRV2.1.1 grouping and intervention51 SPF Balb/c newborn mice were randomly divided into A,B,and C groups(n=17).Mice in group A,B and C were intraperitoneally injected with 30ul 105 PFU/ml,106 PFU/ml and 107 PFU/ml RRV within 24 hours after birth respectively.The number of newborn mice with yellow staining was recorded and the proportion of yellow staining was calculated.The time of yellow staining and survival days was recorded,and the survival curve was drawn.2.2 intervention of jiawei yinchensini decoction on newborn mice with BA model2.2.1 grouping60 newborn SPF Balb/c mice were randomly divided into 6 groups,including the blank group,the model group,the high dose group of jiawei yinchensini decoction,the medium dose group of jiawei yinchensini decoction,the low dose group of jiawei yinchensini decoction and the ursodeoxycholic acid group(n=10).2.2.2 treatmentThe blank group were fed normally,and the rest were intraperitoneally injected with 30ul 106 PFU/ml rhesus rotavirus within 24h after birth.Neonatal rats were given gastric gavage once a day from the 5th day to the 12th day after birth.The high-dose group,medium-dose group and low-dose group of jiawei yinchensini decoction were treated with jiawei yinchensini decoction of 3.47 g/(kg·d),1.73 g/(kg·d)and 0.87 g/(kg·d),the ursodeoxycholic acid group were treated with ursodeoxycholic acid of 1.08 mg/(kg·d),and the model group were treated with 10 ml/(kg·d)0.9%NaCl.2.2.3 the research content2.2.3.1 The body weights of mice were continuously recorded and the change curve of body weight was drawn from the 5th to 12th day and the change of yellow staining in mice were observed.2.2.3.2 Neonatal mice were anesthetized by intraperitoneal injection of 10%chloral hydrate at 3.5 ml/kg on the 12th day after birth.Liver tissues were collected,liver mass was recorded,and liver index(liver index=liver mass/body weight x 100%)was calculated.2.2.3.3 HE staining was performed on the liver tissues of each group.2.3.4 The relative expression of T?RI mRNA,smad3 mRNA,collagen ? mRNA and collagen ?mRNA were detected by RT-qPCR.2.2.3.5 The relative expression of MMP9 mRNA,TIMP1 mRNA and TIMP2 mRNA were detected by RT-qPCR.2.2.3.6 The relative expression of T?RI protein,smad3 protein,collagen? protein and collagen ? protein were detected by western blotting.2.2.3.7 The relative expression of MMP9 protein,TIMP1 protein and TIMP2 protein were detected by western blotting.Results1.clinical research1.1 33 cases of children were included into this research.The ratio of male to female was 5:3.1.2 The frequency of the symptoms were lamps in the abdomen(33/33),lusterless complexion(32/33),abdominal distension(26/33),yellow staining(23/33),growth retardation(23/33),exposure of abdominal vein(20/33),pale yellow stool(8/33),anorexia(4/33),undertemperature of hand and foot(3/33).1.3 The complications were liver dysfunction(31/33),fibrosis(27/33),cholangitis(23/33),anemia(20/33),splenomegaly(10/33),umbilical hernia(3/33),cirrhosis(2/33),malnutrition(1/33),hypersplenism(1/33),thrombocytopenia(1/33),and sepsis(1/33).1.4 33 prescriptions for postoperative biliary atresia were statistically-analyzed.A total of 67 traditional Chinese medicines were involved in these prescriptions.27 of them had a frequency over 5,and the top 20 were herba artemisiae,licorice,costustoot,atratylodes rhizome,ligusticum wallichii,poria,grifola,aconitum napellus,magnolia officinalis,giant knotweed,rhubarb,gardenia,medicated leaven,vinegar turtle shell,dried orange peel,alisma,dandelion,radix pseudostellariae,red paeony root,dried ginger.1.5 Four properties of these drugs were mainly cold and warm and five tastes of them were mainly bitter,bitter and sweet,and they acted on the spleen,stomach and liver channels.1.6 At the support level of 25%and confidence level of 0.9,a total of 11 drug models were obtained,containing 6 traditional Chinese medicines,namely,herba artemisiae,ligusticum wallichii,poria,atractylodes rhizome,costustoot and licorice.1.7 Correlation degree 8 and penalty degree 2 were selected for cluster analysis,and 6 new prescription combinations were extracted.New prescription 1:scutellaria baicalensis,grifola,radix bupleuri,cortex phellodendri chinensis.New prescription 2:plantain seed,plantain herb,atractylodes macrocephala,dried ginger.New prescription 3:spina gleditsiae,dandelion,wine cornus,lysimachia christinae hance.New prescription 4:aconitum napellus,poria,radix pseudostellariae,honeysuckle.New prescription 5:dandelion,radix pseudostellariae,rhubarb,dried orange peel.New prescription 6:gardenia,radix pseudostellariae,giant knotweed,rhubarb.2.experimental research2.1 the rate of yellowing staining in BA model constructed by RRV with different titersThe rate of yellow staining of mice was 76.47%in group A,88.23%in group B and 100%in group C.The rate of yellowing staining in group C was higher than that of group A(P<0.01).2.2 the emergence time of yellow staining and survival time of BA model constructed by RRV with different titersCompared with group A,the appearances of yellow staining were earlier in group B and C(P<0.01).Compared with group B,there was no significant difference in the appearance of yellow staining in group C(P>0.05).Compared with group A,the survival rates in group B and C decreased(P<0.01).Compared with group B,there was a lower survival rate in group C(P<0.01).According to the survival curve analysis,the median survival time of BA positive mice in group A,B and C were about 14 days,13 days and 10 days.2.3 the effect of jiawei yinchensini decoction on symptoms of mice with biliary atresiaCompared with the blank group,mice in the model group of biliary atresia showed yellow staining and slow or stagnant weight growth.Compared with the model group,the weight of mice in the high,medium and low dose groups of jiawei yinchensini decoction and the ursodeoxycholic acid group increased in different degrees and the yellow staining decreased.2.4 the effect of jiawei yinchensini decoction on liver quality and liver index of mice with biliary atresiaCompared with the blank group,the liver quality in the model group of biliary atresia was decreased(P<0.01).Compared with the model group,the liver quality in the high and medium dose groups of jiawei yinchensinidecoction were significantly increased(P<0.01),while the liver quality in the low dose group of jiawei yinchensini decoction and the ursodeoxycholic acid group were not significantly increased(P>0.05).Compared with the blank group,the liver index in the model group was increased(P<0.01).Compared with the model group,the liver indexes in the high,medium and low dose groups of jiawei yinchensini decoction and the ursodeoxycholic acid group weresignificantly decreased(P<0.01).2.5 the effect of jiawei yinchensini decoction on HE staining of liver tissue in BA miceCompared with the blank group,a large number of inflammatory cells were clustered around the portal area in the model group of biliary atresia.Compared with the model group,the inflammatory cells in the portal area were decreased in different degrees in the high,medium and low dose groups of jiawei yinchensini decoction and the ursodeoxycholic acid group.2.6 the effect of jiawei yinchensini decoction on the relative expression of T?R? mRNA and T?R? protein in the liver tissue of mice with BACompared with the blank group,the relative expressions of T?R? mRNA and T?R? protein in the model group of biliary atresia were significantly increased(P<0.01).Compared with the model group,the relative expression of T?R? mRNA and T?R? protein in the high,medium and low dose group groups of jiawei yinchensini decoction and the ursodeoxycholic acid group were decreased(P<0.05 or P<0.01).2.7 the effect of jiawei yinchensini decoction on the relative expression of smad3 mRNA and smad3 protein in the liver tissue of mice with BACompared with the blank group,the relative expressions of smad3 mRNA and smad3 protein in the model group of biliary atresia were increased(P<0.05).Compared with the model group,the relative expressions of smad3 mRNA and smad3 protein in the high,medium and low dose groups of jiawei yinchensini decoction were decreased(P<0.05 or P<0.01),and the relative expressions of smad3 protein in the ursodeoxycholic acid group were decreased(P<0.05)while the relative expressions of smad3 mRNA in the ursodeoxycholic acid group were decreased without statistical significance(P<0.05).2.8 the effect of jiawei yinchensini decoction on the relative expression of collagen ? mRNA and collagen ? protein in the liver tissue of mice with BACompared with the blank group,the relative expressions of collagen I mRNA and collagen ? protein in the model group of biliary atresia were increased without statistical significance(P>0.05).Compared with model group,the relative expressions of collagen ? mRNA and collagen I protein in the high,medium and low dose groups of jiawei yinchensini decoction and the ursodeoxycholic acid group were reduced without statistical significance(P>0.05).2.9 the effect of jiawei yinchensini decoction on the relative expression of collagen ? mRNA and collagen ? protein in the liver tissue of mice with BACompared with the blank group,the relative expressions of collagen ?mRNA and collagen ? protein in the model group of biliary atresia were increased without statistical significance(P>0.05).Compared with model group,the relative expressions of collagen ? mRNA and collagen ? protein in the high,medium and low dose groups and the ursodeoxycholic acid group were reduced without statistical significance(P>0.05).2.10 the effect of jiawei yinchensini decoction on the relative expression of MMP9 mRNA and MMP9 protein in the liver tissue of mice with BACompared with the blank group,the relative expressions of MMP9 mRNA were significantly increased in the model group of biliary atresia(P<0.01),while the relative expressions of MMP9 protein were increased without statistical significance(P>0.05).Compared with model group,the relative expressions of MMP9 mRNA and MMP9 protein in the high,medium and low dose groups were reduced without statistical significance(P>0.05).2.11 the effect of jiawei yinchensini decoction on the relative expression of TIMP1 mRNA and TIMP1 protein in the liver tissue of mice with BACompared with the blank group,the relative expressions of TIMP1 mRNA and TIMP1 protein in the model group of biliary atresia were significantly increased(P<0.01).Compared with model group,the relative expressions of TIMP1 mRNA and TIMP1 protein in the low dose group of jiawei yinchensini decoction and the ursodeoxycholic acid group were significantly lower(P<0.05 or P<0.01),the relative expressions of TIMP1 mRNA and TIMP1 protein in the medium dose group of jiawei yinchensini decoction were reduced without statistical significance(P>0.05).In the high dose group,the relative expressions of TIMP1 mRNA were reduced without statistical significance CP>0.05)while the relative expressions of TIMP1 protein were reduced(P<0.05).2.12 the effect of jiawei yinchensini decoction on the relative expression of TIMP2 mRNA and TIMP2 protein in the liver tissue of mice with BACompared with the blank group,the relative expressions of TIMP2 mRNA and TIMP2 protein in the model group were increased without statistical significance(P>0.05).Compared with model group,the relative expressions of TIMP2 mRNA and TIMP2 protein in the high,medium and low dose groups of jiawei yinchensini decoction and the ursodeoxycholic acid group were reduced without statistical significance(P>0.05).Conclusion1.clinical study(1)Combined with the postoperative symptoms and complications of BA,biliary atresia can be categorized as "jaundice" or "abdominal mass" from the point of view of traditional Chinese medicine.Lamps in the abdomen and exposure of abdominal vein suggested the existence of venous stasis.Lusterless complexion indicated the existence of deficiency of qi and blood and spleen.Abdominal distension and anorexia suggested the existence of water transport barriers.Yellow staining could be distinguished by cold wet or dampness hot which accumulate liver and the gall bladder.Growth retardation and cold hands and feet indicated congenital deficiency of kidney qi and deficiency of kidney Yang.The pathogenesis of this disease is a mixture of deficiency and solid,considering the congenital deficiency of spleen and kidney,and the combination of "blood stasis","stagnation" and "heat",causing the disease.In terms of treatment,it is necessary to strengthen spleen,warm Yang,benefit dampness,change dampness,remove yellows,promote blood circulation and remove blood stasis,and regulate qi.(2)The most common complication is liver dysfunction.Postoperative hepatic fibrosis and recurrent cholangitis have a high incidence,which seriously affected the growth and development of children with BA.This indicated that BA children had hepatobiliary dampness-heat and bile extravasation,which should be treated with heat-clearing and dampness-relieving therapy.(3)Four properties of these drugs in these 33 prescriptions were mainly cold and warm and five tastes of them were mainly bitter,bitter and sweet,and they act on the spleen,stomach and liver channels.This reflects li's pediatric academic thought of"cold and warm combination" oin the treatment of biliary attrition.(4)In this study,the traditional Chinese medicine prescriptions of 33 children were analyzed.The basic prescriptions for the treatment of children with biliary atresia after surgery were summarized the top 20 medicines:Jiawei Yinchensini decoction,Yinchenhao decoction,Yigong powder,wuling powder and so on,which laid a foundation for the follow-up mechanism research on the treatment of biliary atresia with traditional Chinese medicine.(5)At the support level of 25%and confidence level of 0.9,a total of 11 drug models were obtained,containing 6 traditional Chinese medicines:herba artemisiae,ligusticum wallichii,poria,atractylodes rhizome,costustoot and licorice.Here herba artemisiae was used to let yellow back,ligusticum wallichii was used to invigorate the circulation of blood,poria was used to transport the spleen wet,atractylodis rhizoma was used to dry the wet,costustoot was used to run qi and licorice was used to blends the herbs.This reflects the li' s department of pediatrics' academic thought of "tonification and purgation in combination"in the treatment of biliary atresia,to invigorate spleen for eliminating dampness and regulate qi to disperse stagnation,and to make huang degrade with dampness.(6)When the correlation degree was 8 and the penalty degree was 2,6 new prescription combinations were extracted.In the six new formulae combinations extracted,the drugs were mostly cold and warm,which also reflected the academic thought of "warming up simultaneously,attacking and supplementing simultaneously" in the treatment of BA Kasai in lingnan pediatric department,and provided a new idea for the treatment of liver and gallbladder damp-heat syndrome,spleen and kidney Yang deficiency syndrome,liver and kidney Yin deficiency syndrome and so on in children with BA.2.experimental research(1)The higher the titer of RRV was,the earlier the yellow staining of BA positive mice appeared,and the higher the yellow staining rate was.That indicated a higher success rate of the modeling,but a shorter survival time.(2)Intraperitoneal injection of 30ul 106 PFU/ml RRV into Balb/c newborn mice within 24 hours after birth can successfully construct the BA model with a good reproducibility and long survival time.(3)Jiawei yinchensini decoction can increase the weight and liver quality,promote the growth and development,reduce the liver index,alleviate the aggregation of inflammatory cells in the portal area and reduce the liver inflammation of mice.(4)Jiawei yinchensini decoction may delay the process of biliary atresia by reducing T?R?,decreasing the combination of TGF-?1 and T?R?,inhibiting the phosphorylation process of T?R?and reducing the secretion of smad3.(5)Due to the short survival time of BA mice,collagen ? and collagen ?deposition and efficacy of jiawei yinchensini decoction were not observed.(6)Jiawei yinchensini decoction may reduce the binding of TIMP1 to MMP9 active sites by inhibiting the relative expression of TIMP1,and increase the active MMP9 indirectly,which might promote the degradation of ECM. |
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