Correlation Analysis Of TCM Syndromes Of Hepatitis B Cirrhosis And Study On The Mechanism Of Action Of Yiqihexue Prescription In Treating Liver Cirrhosi

Background:Liver cirrhosis is a pathological stage characterized by diffuse fibrosis of the liver,pseudolobule formation,and proliferation of blood vessels inside and outside the liver.It is characterized by high mortality and high incidence rate.Traditional Chinese medicine has its unique advantages in treating liver cirrhosis.The types of traditional Chinese medicine syndromes in liver cirrhosis are very complex.Professor Guan Youbo believes that liver cirrhosis is based on qi deficiency and blood stagnation,and the Yiqi Hexue formula was formulated by the department of Hepatology,Beijing Hospital of Traditional Chinese Medicine under the academic thought of Professor Guan Youbo’s"Syndrome differentiation of Qi and blood".It has been proven in clinical practice to be effective in treating liver cirrhosis,but its mechanism against liver cirrhosis still needs further exploration.Objective:1.To explore the distribution of traditional Chinese medicine(TCM)syndrome types of liver cirrhosis and the correlation between TCM syndrome types and objective indicators,understand the distribution characteristics of TCM syndrome types of hepatitis B cirrhosis,and provide objective basis for TCM syndrome differentiation.2.Identify the main active ingredients of Yiqi Hexue formula,analyze the pharmacological substance basis of Yiqi Hexue formula in treating liver cirrhosis,and predict the potential targets and corresponding pathways of these compounds in treating liver cirrhosis.3.Explore the therapeutic effect of Yiqi Hexue formula on carbon tetrachloride(CCl4)induced liver cirrhosis in rats,and reveal its potential molecular mechanisms.Method:1.Collect patients with hepatitis B cirrhosis from the department of Hepatology,Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,including their name,gender,age,main symptoms,tongue and pulse,TCM syndrome classification,Child Pugh grading,liver cirrhosis staging,liver function(AST,ALT,GGT,ALP,CHE,ALB,TBIL),PT,PTA,ascites,and hepatic encephalopathy data,Statistical analysis was carried out through SPSS26.0 statistical software to observe the overall distribution of TCM syndrome types in patients with hepatitis B cirrhosis,and to statistically analyze the correlation between TCM syndrome types and general conditions(gender,age),Child-Pugh classification,cirrhosis stage,liver function,coagulation function and other objective indicators.2.Qualitative analysis of compounds in Yiqi Hexue formula was carried out using ultraperformance liquid chromatography-quadrupole-Exactive Orbitrap-mass spectrometry(UPLC-Q-Exactive Orbitrap-MS),All data were collected and analyzed using Xcalibur 4.1 software,and further confirm the qualitative chemical composition based on the relative retention time and fragment ion information provided in the literature.Using the TCMSP platform and the Swiss Target Prediction database,target search was conducted on each compound identified in the Yiqi Hexue formula.Search for liver cirrhosis related genes from GeneCard,PharmGkb,OMIM,DrugBank,TTD databases.The intersection of the two is the potential target of Yiqi Hexue formula in treating liver cirrhosis.Construct a regulatory network diagram of "drug active ingredients-disease targets",construct a protein interaction network,and screen core genes.And through KEGG pathway enrichment analysis,predict the potential mechanism of action of Yiqi Hexue formula in treating liver cirrhosis.3.69 SPF grade SD male rats were selected,and 9 rats were randomly selected as the normal group.The remaining 60 model group rats were injected intraperitoneally with a dose of 2 ml/kg of 50%CC14 solution(solvent:olive oil)for 2 weeks and 3 ml/kg for 4 weeks,twice a week.The normal group rats were injected intraperitoneally with an equal volume of olive oil.After a total of 6 weeks of modeling,3 rats were randomly selected from the modeling group and 3 rats from the normal group.After successful modeling was evaluated through pathological examination,they were randomly divided into the model group,the low dose group of Yiqi Hexue formula,the medium dose group of Yiqi Hexue formula,the high dose group of Yiqi Hexue formula,and the Fuzheng Huayu group,with 8 rats in each group.The low,medium,and high dose groups of Yiqi Hexue formula were given 0.4305 g/100 g,0.861 g/100 g,and 1.722 g/100 g by gavage,respectively.The Fuzheng Huayu group was given 0.04725 g/100 g by gavage,while the model group and normal group were given physiological saline by gavage,with a dosage of 1 ml/100 g.All rats were gavaged once a day for 4 consecutive weeks.At the end of the experiment,the liver of each group of rats was taken and serum was collected.HE staining and Masson staining were performed on the liver tissues of rats in each group to observe pathological changes.The levels of ALT,AST,TBIL,DBIL,ALP,and GGT in the serum samples of each group of rats were detected using an automatic serum biochemical analyzer.The levels of HA,LN,IV-C,and PCⅢ were detected using ELISA;Alkaline hydrolysis method was used to determine the content of liver hydroxyproline(HYP);qRT-PCR detection of rat liver tissue α-SMA,COL-Ⅰ,COL-ⅢmRNA expression;Using Western blot assay to detect the presence of α-SMA protein expression and p-ERK1/2,ERK1/2,p-JNK,JNK,p-p38,and p38 protein expression;Using immunohistochemistry to detect the expression of p-ERK1/2,p-JNK,and p-p38 in liver tissue.Result:1.A total of 184 patients who met the criteria were included,including 80 females,104 males,42 decompensated patients,and 142 compensatory patients,with an average age of 56.63±10.25 years.Liver stagnation and spleen deficiency,qi deficiency and blood stasis are the most common syndrome types in patients with hepatitis B cirrhosis in the outpatient department of hepatology.This study mainly discusses 173 cases of liver depression and spleen deficiency syndrome,qi deficiency and blood stasis syndrome,dampheat accumulation syndrome,water dampness internal obstruction syndrome,and qi stagnation and blood stasis syndrome,The results show that in general,there is no difference in the distribution of gender and age among different syndrome types(P>0.05);There was no significant difference in the distribution of Child-Pugh among the various syndrome types of hepatitis B cirrhosis patients(P>0.05).From the perspective of liver cirrhosis staging,the liver depression and spleen deficiency syndrome was more common in the compensatory period,and the qi deficiency and blood stasis syndrome was more common in the decompensated period.The liver cirrhosis staging had differences among the various syndrome types(P<0.05);In terms of liver function,there is a statistically significant difference in ALT among different syndrome types(P<0.05),with liver depression and spleen deficiency syndrome>qi deficiency and blood stasis syndrome.Among different syndrome types,the difference in ALP also has statistical significance(P<0.05),with liver depression and spleen deficiency syndrome>damp-heat accumulation syndrome.There was no statistically significant difference in AST,ALB,TBIL,GGT,and CHE among different syndrome types(P>0.05);In terms of coagulation function,there are differences in the distribution of PT and PTA among different syndrome types(P<0.05).The maximum value of PT is qi deficiency and blood stasis syndrome,with qi deficiency and blood stasis syndrome>dampness heat accumulation syndrome.The minimum value of PTA is qi deficiency and blood stasis syndrome,with damp-heat accumulation syndrome>qi deficiency and blood stasis syndrome.2.Using UPLC-Q-Exactive Orbitrap-MS,32 components were identified in Yiqi Hexue formula,including gallic acid,paeoniflorin,paeonol,calycosin,and formononetin.Using network pharmacology,210 candidate targets were identified for the treatment of liver cirrhosis with Yiqi Hexue formula.After screening,19 core genes were obtained,including PPARA,SRC,JUN,MAPK14,MAPK1,PIK3CA,MAPK3,TP53,RELA,IL2,NFKBIA,AKT1,etc,The KEGG enrichment analysis results show that the mechanism of action of Yiqi Hexue formula in treating liver cirrhosis is mainly related to signal pathways such as PI3K-Akt,MAPK,IL-17,VEGF,TNF,etc.3.After 4 weeks of gavage,the general condition of rats in each treatment group improved and their weight increased.The results of HE and Masson staining show that in rats treated with CC14,severe damage to the structure of the liver lobe was marked,swelling and irregular arrangement of hepatocytes,extensive steatosis and necrosis of hepatocytes,inflammatory cell infiltration around the portal area and central vein,appearance of fibrous septa and collagen deposited to form pseudolobules.Compared to the model group,inflammatory cell infiltration,hepatocyte necrosis,and steatosis were reduced,and collagen fibers were significantly reduced in each treatment group.Especially in the middle and high dose Yiqi Hexue Formula group and the Fuzheng Huayu group,the improvement of inflammatory cell infiltration and collagen deposition was more obvious.Compared with the model group,there were statistically significant differences in the middle and high doses of Yiqi Hexue formula and the Fuzheng Huayu Group(P<0.05,P<0.01).Compared with the model group,the serum levels of ALT,ALP,GGT,TBIL,and DBIL in the low,medium,and high dose groups of Yiqi Hexue Formula and the Fuzheng Huayu Group were significantly reduced(P<0.05,P<0.01,P<0.001),while the AST levels in the medium,high dose groups of Yiqi Hexue Formula and the Fuzheng Huayu Group were significantly reduced(P<0.001).Compared with the model group,the serum HA and PCⅢ of rats in the low,medium,and high dose groups of Yiqi Hexue Formula and the Fuzheng Huayu Group were significantly reduced(P<0.01,P<0.001).The serum LN of rats in the medium and high dose groups of Yiqi Hexue Formula and the Fuzheng Huayu Group was significantly reduced(P<0.05,P<0.01),and the IV-C of rats in the medium and high dose groups of Yiqi Hexue Formula was significantly reduced(P<0.05).Compared with the model group,the treatment groups of Yiqi Hexue Formula and the Fuzheng Huayu group showed a statistically significant decrease in HYP(P<0.001).Compared with the model group,the treatment groups of Yiqi Hexue Formula,as well as the Fuzheng Huayu group,showed significant inhibition expression of α-SMA and COL-Ⅰ,COL-Ⅲ mRNA,and αSMA protein expression(P<0.001).The results of protein quantitative analysis showed that compared with the model group,the p-ERK/ERK protein expression ratio in the liver tissue of rats in the high-dose group of Yiqi Hexue Formula was significantly reduced(P<0.01),while the p-JNK/JNK,p-p38/p38 protein expression ratio in the liver tissue of rats in the various dose groups of Yiqi Hexue Formula and the Fuzheng Huayu group was significantly reduced(P<0.05,P<0.01,P<0.001).Immunohistochemical analysis showed that compared to the normal group,the positive expression of p-ERK1/2,p-JNK,and p-p38 in the model group was significantly enhanced(P<0.001),and the positive cells were mainly located in the portal area,the fibrous septum and around the central lobular vein.However,compared to the model group,the protein expressions of p-ERK1/2,p-JNK,and p-p38 were significantly attenuated after treatment with Yiqi Hexue Formula and Fuzheng Huayu(P<0.01,P<0.001)Conclusion:1.Liver stagnation and spleen deficiency,qi deficiency and blood stasis are the most common syndromes in patients with hepatitis B cirrhosis in the outpatient department of hepatology.The distribution of syndrome types suggests that the syndrome of this disease is mainly characterized by liver depression,dampness-heat,and blood stasis,while deficiency syndrome is mainly characterized by qi deficiency.In the compensatory period,liver depression and spleen deficiency syndrome are more common,while in the decompensated period,qi deficiency and blood stasis syndrome are more common.There may be a certain correlation between liver function,coagulation routine,and syndrome types.2.There are various chemical components in the Yiqi Hexue Formula.Gallic acid,paeoniflorin,paeonol,calycosin,and formononetin may be the main components that exert anti liver cirrhosis effects.The characteristic of Yiqi Hexue Formula is that it acts on multiple targets with multiple components and exerts anti liver cirrhosis effects through multiple pathways.3.Yiqi Hexue Formula can improve the body weight and other general conditions of carbon tetrachloride induced liver cirrhosis rats,reduce its histological performance,improve liver function in rats,reduce the levels of fibrosis markers in serum and the content of HYP in liver tissue,reduce the expression of α-SMA and type Ⅰ and Ⅲ collagen.Yiqi Hexue Formula may exert therapeutic effects on liver cirrhosis by inhibiting the MAPK signaling pathway.