Predicting Target Genes of San-Huang-Chai-Zhu Formula in Treating ANIT-Induced Acute Intrahepatic Cholestasis Rat Model via Bioinformatics Analysis Combined with Experimental Validation.

Jiaming Yao,Zhiyun Chen,Xi Chen,Junbin Yan,Beihui He,Jinting Wu,Jianshun Yu,Priscila Souza

doi:10.1155/2021/5320445

Abstract

Background San-Huang-Chai-Zhu formula (SHCZF) has been used to improve cholestasis for many years. This study aims to predict the possible gene targets of SHCZF in treating acute intrahepatic cholestasis (AIC) in rats. Materials and Methods Eighteen SD rats were randomly assigned to the normal group, ANIT group, and ANIT + SHCZF group. Alpha-naphthylisothiocyanate (ANIT) was used to induce AIC. Serum biochemical indexes were detected in each group. After treatment, the livers were collected and used to extract RNA. The library was constructed by TruSeq RNA, sequenced by Illumina, and analyzed by various bioinformatics methods. qRT-PCR was used to verify the target genes related to the efficacy of SHCZF. Results Serum ALT, AST, ALP, and TBIL were significantly higher in the ANIT group than in the normal group. Serum ALT and AST levels in the ANIT + SHCZF group were substantially lower than those in the ANIT group. A total of 354 intersected genes were screened by expression level correlation and PCA analysis, GO and KEGG pathway enrichment analysis, and WGCNA and STEM analysis. Then, 4 overlapping genes were found by pathway/BP/gene network construction. SHCZF reversed the downregulation of expression of CYP4A1 and HACL1 and the upregulation of expression of DBI and F11R induced by ANIT. In addition, the qRT-PCR result showed that mRNA expression of CYP4A1, HACL1, and F11R genes in the liver was consistent with the prediction result of bioinformatics analysis. Conclusion CYP4A1, HACL1, and F11R are genes related to the occurrence of ANIT-induced AIC in rats and may be considered as targets of SHCZF for the treatment of AIC.

Highlights

Intrahepatic cholestasis is a primary hepatocellular disorder associated with abnormal formation, secretion, and/or excretion of bile that is asymptomatic in the early stage
E serum levels of ALT and AST in the ANIT + San-Huang-Chai-Zhu formula (SHCZF) group were significantly lower compared to the ANIT group (p < 0.01), while there was no significant difference in ALP and TBIL levels (p > 0.05) (Figure 3). ese findings suggested that SHCZF has a significant protective effect on acute intrahepatic cholestasis (AIC)
After filtering out the unqualified low-quality data, 231,585,315 high-quality sequences were obtained, accounting for 99.15% of the total. en, using TopHat software algorithm, clean reads were compared to the reference genome; the results showed that the sequences that could be compared to the genome accounted for 85.97% of the total clean reads, 77.93% of which had unique location

Summary

Introduction

Intrahepatic cholestasis is a primary hepatocellular disorder associated with abnormal formation, secretion, and/or excretion of bile that is asymptomatic in the early stage. Its progression is manifested as jaundice, weakness, dark urine color, and skin pruritus. If left untreated, it may cause cirrhosis, portal hypertension, and liver failure [1]. No diagnostic criteria for intrahepatic cholestasis have been proposed. Cao et al [2] studied the prevalence of cholestasis in the 4660 inpatients with chronic liver disease in Shanghai, China, revealing that ALP level higher than 1.5 times of the upper limit of normal value (ULN) and GGT level higher than 3 times of ULN could be used as diagnostic standards for the disease. Is study aims to predict the possible gene targets of SHCZF in treating acute intrahepatic cholestasis (AIC) in rats. The livers were collected and used to extract RNA

Methods

Results

Discussion

Conclusion