# Serum TGF-β1 and VEGF Levels Reflect the Liver Hardness and Function in Children with Biliary Atresia

Authors: Dongzhe Chang; Xianjie Geng; Liang Zhou; Guangjun Hou
Journal: Computational and Mathematical Methods in Medicine
 (2022)

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## Abstract

Objective. This study further explored the wind direction correlation analysis between serum levels of TGF-β1 and VEGF and liver function assessment in children with biliary atresia. Methods. A total of 62 children with biliary atresia (BA) who received surgical treatment in our hospital from October 2020 to October 2021 were selected as the research objects (BA group), and 50 normal healthy children who received routine physical examination in our hospital during the same period were selected as blank control group. Outcome measures included postoperative total bilirubin levels and conjugality of enrolled patients. Bilirubin level, unbound bilirubin level, serum transforming growth factor-beta-1 (TGF-β1), vascular endothelial growth factor (VEGF), liver function indicators albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and other observation indicators were included. All data in this study were collected and analyzed by SPSS 23.0 software, and t-test was performed. Results. The serum levels of TGF-β1, VEGF, ALT, AST, GGT, and liver hardness were significantly higher in children with jaundice than those without jaundice, and the serum ALB level was significantly lower than that in children without jaundice (P<0.05). The levels of TGF-β1 and VEGF in BA group were positively correlated with the levels of ALT, AST, GGT, and liver hardness (P<0.05) but negatively correlated with the level of ALB (P<0.05). Conclusion. The levels of serum TGF-β1 and VEGF in children with biliary atresia have a certain risk correlation with liver function damage, which will become a research focus on the mechanism of liver fibrosis in the diagnosis and treatment of biliary atresia in children.


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## Body

## 1. Introduction

Biliary atresia (BA) is a severe neonatal disease with unknown etiology characterized by progressive inflammation of the bile duct and biliary fibrous obstruction [1]. If BA is not timely intervention, most children will die due to liver failure at the age of 2 years. Surgical intervention is the only effective treatment [2]. BA is one of the common causes of neonatal obstructive jaundice, and its pathogenesis is not fully defined [3–5]. There are significant regional and ethnic differences in BA incidence, with higher rates in Asian countries than in European and American countries [6]. Biliary atresia refers to a series of serious malformations characterized by cholestatic liver injury due to progressive inflammatory or fibrotic atresia of the intrahepatic bile ducts, with persistent jaundice as the main clinical manifestation [7]. The disease progresses rapidly and can cause death within 2 years of age. Kasai surgery in early stage is the preferred treatment for this disease, but due to the lack of specific indications, it is difficult to prompt diagnosis based on clinical manifestations [8]. In order to make up for this deficiency, researchers based on the characteristics of biliary atresia lead to more obvious liver fibrosis. Shear wave elastography was used to measure and compare the liver cirrhosis of biliary atresia and other pathological jaundice, and the results of hardness difference support the identification of biliary atresia [9]. In theory, if the influence of individual baseline biochemical level can be fully considered in the application, it is expected to improve the practical diagnostic value of elastography in different populations [10].It has been reported that few cytokines and growth factors, including TGF-β1, can increase in VEGF protein levels and induce its secretion in different types of cells [11]. Our previous study has demonstrated that TGF-β1 can regulate steroidogenesis, cell proliferation, and differentiation in hGL cells [12]. A previous study showed that TGF-β1 increases the secretion of VEGF and stimulates angiogenic activity in rat granulosa cells [13]. However, whether the same effect is true for human granulosa cells remains unknown. In order to avoid bias of disease progression, the interval between blood sample collection and elastography examination was compressed to within 3 days on the premise of ensuring medical arrangement [14]. Biliary atresia (BA) is the most common cause of obstructive jaundice in neonates, with an incidence of approximately 1/10000 [15]. The pathological feature of BA is that the lesion involves the intrahepatic bile duct system, resulting in progressive bile duct destruction [16]. Successful hilar jejunostomy (Kasai) allows bile to be drained and some children to survive [17]. However, due to the occurrence of progressive liver fibrosis, cholestatic cirrhosis, and portal hypertension, most children must choose liver transplantation. Progressive liver fibrosis is a major constraint on Kasai’s surgical results which show that in cervical, preventing the progression of liver fibrosis will significantly improve the outcome of Kasai surgery, and the transforming growth factor-β1 (TGF-β1) protein regulates cell growth [18].In order to explore the expression level and diagnostic value of TGF-1 and VEGF in BA hepatic fibrosis, the general clinical data of 62 children with biliary atresia admitted to our hospital from March 2016 to 83 Months 2019 were collected, and TGF-1 and VEGF were detected and analyzed, to explore the expression level and diagnostic value of TGF-1 and VEGF in biliary atresia liver (BA) fibrosis.


## 2. Materials and Methods

### 2.1. General Clinical Features

The inclusion criteria were as follows: (1) BA was diagnosed by clinical and pathological sections, (2) complete clinical data and samples, (3) mild hepatic fibrosis was judged by liver HE staining, (4) the postoperative recovery was good without postoperative complications, and (5) the informed consent of the family members of the children was obtained.Exclusion criteria were as follows: (1) combined with other hepatobiliary diseases, (2) combined with other congenital diseases, and (3) liver transplantation was received within 2 months after operation. According to the level of total bilirubin, BA group was divided into children with jaundice (totalbilirubin≥34.2 μmol/L) and children without jaundice (total bilirubin<34.2 μmol/L).Another 50 healthy children who underwent physical examination in our hospital in the same period were taken as the control group. There were 35 males and 27 females in BA group, aged from 5 to 18 months, with an average of12.47±2.28 months. There were 28 males and 22 females in the control group, aged from 5 to 20 months, with an average of 13.11±2.94 months. There was no significant difference in gender and age between BA group and control group (P>0.05). All patients had the written consent explained to them. All patients have signed written informed consent. This study was approved by the medical ethics committee of the Hospital of Zhengzhou University.


### 2.2. Detection of Serum Indicators

Children in BA group collected 3 ml of fasting venous blood 2 months after operation, and children in control group collected 3 ml of fasting venous blood during physical examination. After standing for 30 minutes, 3000 revolutions/heart separation for 10 minutes, serum was separated. TGF was detected by ELISA kit. The ELISA kit was purchased from Beyotime Biotechnology Company, and the operation was carried out according to the instructions of the kit.


### 2.3. Detection of Liver Hardness Value

The children in the BA group were tested for liver hardness at 2 months after operation, and the children in the control group were tested for liver hardness at physical examination. The detection method was instantaneous elastic imaging. The elastic ultrasonic probe was placed in the axillary midline on the right side of the body and recorded continuously. It was successfully tested for 10 times, and the median number was taken as the liver hardness value.


### 2.4. Statistical Analysis

Load extension packages (RMS, RMDA, pROC, and resources election) use R Software 3.61 to analyze the data. Since the measurement data did not conform to the normal distribution, they were represented byM (P25 and P75), and the difference between groups was compared by Mann-Whitney U test. The counting data were represented by example (ratio), and the differences between groups were compared by Pearson’s chi-square test. Due to collinearity among the indicators, according to the data of the training set, the LASSO regression was used to include liver hardness measurements, blood biochemical results, and epidemiological characteristics as independent variables and whether biliary atresia was defined as dependent variables. The 10-fold cross validation method was used to test the fitting results of all the regular penalty terms. The regularization parameter (λ) of the person with minimum deviation (binomial deviance) was calculated, and the simplest model corresponding to one standard error range of the parameter was confirmed, and its construction indexes were screened. Then, the diagnostic model was reconstructed by the odds ratio (OR) of binary logistic regression. The cut-off value was defined by maximum approximation index, and sensitivity, specificity, and corresponding 95% confidence interval (95% CI) were refined. The differentiation degree of the model was evaluated by area under ROC curve (AUC), and the calibration degree was evaluated by Hosmer-Lemeshow test and calibration curve. Statistical graphs were drawn by R Software except ROC curve using GraphPad Prism 8.0. P<0.05 was considered as statistically significant difference.


## 2.1. General Clinical Features

The inclusion criteria were as follows: (1) BA was diagnosed by clinical and pathological sections, (2) complete clinical data and samples, (3) mild hepatic fibrosis was judged by liver HE staining, (4) the postoperative recovery was good without postoperative complications, and (5) the informed consent of the family members of the children was obtained.Exclusion criteria were as follows: (1) combined with other hepatobiliary diseases, (2) combined with other congenital diseases, and (3) liver transplantation was received within 2 months after operation. According to the level of total bilirubin, BA group was divided into children with jaundice (totalbilirubin≥34.2 μmol/L) and children without jaundice (total bilirubin<34.2 μmol/L).Another 50 healthy children who underwent physical examination in our hospital in the same period were taken as the control group. There were 35 males and 27 females in BA group, aged from 5 to 18 months, with an average of12.47±2.28 months. There were 28 males and 22 females in the control group, aged from 5 to 20 months, with an average of 13.11±2.94 months. There was no significant difference in gender and age between BA group and control group (P>0.05). All patients had the written consent explained to them. All patients have signed written informed consent. This study was approved by the medical ethics committee of the Hospital of Zhengzhou University.


## 2.2. Detection of Serum Indicators

Children in BA group collected 3 ml of fasting venous blood 2 months after operation, and children in control group collected 3 ml of fasting venous blood during physical examination. After standing for 30 minutes, 3000 revolutions/heart separation for 10 minutes, serum was separated. TGF was detected by ELISA kit. The ELISA kit was purchased from Beyotime Biotechnology Company, and the operation was carried out according to the instructions of the kit.


## 2.3. Detection of Liver Hardness Value

The children in the BA group were tested for liver hardness at 2 months after operation, and the children in the control group were tested for liver hardness at physical examination. The detection method was instantaneous elastic imaging. The elastic ultrasonic probe was placed in the axillary midline on the right side of the body and recorded continuously. It was successfully tested for 10 times, and the median number was taken as the liver hardness value.


## 2.4. Statistical Analysis

Load extension packages (RMS, RMDA, pROC, and resources election) use R Software 3.61 to analyze the data. Since the measurement data did not conform to the normal distribution, they were represented byM (P25 and P75), and the difference between groups was compared by Mann-Whitney U test. The counting data were represented by example (ratio), and the differences between groups were compared by Pearson’s chi-square test. Due to collinearity among the indicators, according to the data of the training set, the LASSO regression was used to include liver hardness measurements, blood biochemical results, and epidemiological characteristics as independent variables and whether biliary atresia was defined as dependent variables. The 10-fold cross validation method was used to test the fitting results of all the regular penalty terms. The regularization parameter (λ) of the person with minimum deviation (binomial deviance) was calculated, and the simplest model corresponding to one standard error range of the parameter was confirmed, and its construction indexes were screened. Then, the diagnostic model was reconstructed by the odds ratio (OR) of binary logistic regression. The cut-off value was defined by maximum approximation index, and sensitivity, specificity, and corresponding 95% confidence interval (95% CI) were refined. The differentiation degree of the model was evaluated by area under ROC curve (AUC), and the calibration degree was evaluated by Hosmer-Lemeshow test and calibration curve. Statistical graphs were drawn by R Software except ROC curve using GraphPad Prism 8.0. P<0.05 was considered as statistically significant difference.


## 3. Results

### 3.1. Comparison of Serum Indexes and Liver Hardness

Compared with the control group, serum TGF in BA group, The levels of VEGF, ALT, AST, and GGT increased significantly (P<0.05), but there was no significant difference in serum ALB level (P>0.05) (Figure 1).Figure 1
Comparison of serum indexes and liver hardness values between the two groups.


### 3.2. Comparison of Children with Jaundice and Children without Jaundice

In BA group, there were 24 children with jaundice and 38 children without jaundice. Compared with children without jaundice, serum TGF in children with jaundice, The levels of VEGF, ALT, AST, GGT, and liver hardness increased significantly, while the level of ALB decreased significantly (P<0.05) (Figure 2).Figure 2
Comparison of serum indexes and liver hardness values of children with jaundice and children without jaundice in the BA group.


### 3.3. Serum TGF in BA Group: Correlation between Liver Hardness and Liver Function

Pearson’s correlation analysis showed the serum TGF in BA group. There was a positive correlation between liver hardness and serum VEGF (P<0.05). The level of VEGF was positively correlated with the levels of ALT, AST, and GGT and negatively correlated with the level of ALB (P<0.05) (Table 1).Table 1
Correlation of serum TGF-1 and VEGF with liver hardness values and liver function in the BA group.
IndexLiver hardnessALBALTASTGGTrPrPrPrPrPTGF-β10.3780.004-0.2470.01202840.0140.2840.0040.2540.008VEGF0.2920.010-0.3620.0020.2130.0120.3640.0010.2360.004


## 3.1. Comparison of Serum Indexes and Liver Hardness

Compared with the control group, serum TGF in BA group, The levels of VEGF, ALT, AST, and GGT increased significantly (P<0.05), but there was no significant difference in serum ALB level (P>0.05) (Figure 1).Figure 1
Comparison of serum indexes and liver hardness values between the two groups.


## 3.2. Comparison of Children with Jaundice and Children without Jaundice

In BA group, there were 24 children with jaundice and 38 children without jaundice. Compared with children without jaundice, serum TGF in children with jaundice, The levels of VEGF, ALT, AST, GGT, and liver hardness increased significantly, while the level of ALB decreased significantly (P<0.05) (Figure 2).Figure 2
Comparison of serum indexes and liver hardness values of children with jaundice and children without jaundice in the BA group.


## 3.3. Serum TGF in BA Group: Correlation between Liver Hardness and Liver Function

Pearson’s correlation analysis showed the serum TGF in BA group. There was a positive correlation between liver hardness and serum VEGF (P<0.05). The level of VEGF was positively correlated with the levels of ALT, AST, and GGT and negatively correlated with the level of ALB (P<0.05) (Table 1).Table 1
Correlation of serum TGF-1 and VEGF with liver hardness values and liver function in the BA group.
IndexLiver hardnessALBALTASTGGTrPrPrPrPrPTGF-β10.3780.004-0.2470.01202840.0140.2840.0040.2540.008VEGF0.2920.010-0.3620.0020.2130.0120.3640.0010.2360.004


## 4. Discussion

The surgical effect of biliary atresia depends on the age of the child, the severity of liver fibrosis, and postoperative complications, and progressive liver fibrosis is an important factor affecting the surgical effect [19]. Studying the mechanism of liver fibrosis and preventing liver fibrosis is a major topic for domestic and foreign scholars [20]. In this study, the positive rate of TGF-1 and VEGF in biliary atresia group was higher than that in the control group (P<0.05), and the expression level of TGF-1 was higher than that in biliary dilatation group and the control group (P<0.05). The expression level of TGF-1 in grades I to II patients was significantly increased [21]. Ttgf-1 expression level was significantly decreased in grades III to IV patients (P<0.05), suggesting that TGF-1 expression level was significantly increased in the early stage of fibrosis and significantly decreased in the late stage of TGF-1 [22]. The reason for the decreased late expression is that the transcription of TGF-1 mRNA in BA hepatosclerosis stage was inhibited, its content decreased, and its promoting fibrosis effect weakened. Some scholars believe that during the cirrhosis of children with BA, due to intrahepatic fibrous hyperplasia and blood circulation disorder, TGF-1 and VEGF proteins are greatly reduced, which is consistent with the results of this experiment [23].Conventional ultrasound has been widely used in the evaluation of jaundice in infants due to its unique advantages of noninvasive, simple, and repeatable. In order to improve the diagnostic level of biliary atresia, based on the long-term accumulated clinical experience, previous researchers have summarized the characteristics of biliary atresia represented by gallbladder morphology and hilar triangle abnormality (TC). However, due to the difficulty in timely detection of TC in children in small age groups and low sensitivity in early stage, the actual clinical value of TC has been doubted [24, 25]. In addition, since pathological jaundice without biliary atresia (such as hepatitis syndrome in infants) can also present with poor gallbladder filling, it has been reported that although the diagnostic sensitivity based on abnormal gallbladder morphology is as high as 97.8%, its specificity is only 54.1% [26–28]. Therefore, a considerable number of jaundice cases evaluated by conventional ultrasound still need to rely on radionuclide angiography, gallbladder puncture angiography, and even open exploration to confirm the diagnosis, and the accompanying risk of radiation injury and surgical complications is inevitable. However, this study also has its limitations, such as the incomplete information for all patients included in this retrospective study. Therefore, more prospective clinical studies and mechanical verifications should be performed in the future study.


## 5. Conclusion

The levels of serum TGF-β1 and VEGF in children with biliary atresia have a certain risk correlation with liver function damage, which will become a research focus on the mechanism of liver fibrosis in the diagnosis and treatment of biliary atresia in children.



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