Whole Exome Sequencing To Identify The Pathogenic Genes Of Cholesterol Gallstones And Experimental Study Of Chinese Medicine Monomer To Prevent Cholesterol Gallstones

Cholesterol gallstone is one of the most common digestive diseases.Studies have shown that cholesterol gallstone is a complex metabolic disease with multiple factors.The research on genetic factors-related pathogenic genes has attracted extensive attention.Whole exome sequence(WES),as a new generation of sequencing technology,is a genome analysis technology that uses sequence capture technology to capture and enrich DNA in whole exon region for high throughput sequencing.The target region of WES is the protein-coding region of the genome containing 80% of human genetic pathogenicity,which is highly sensitive to common and rare mutations and able to find most disease-related mutations in the exon region.The whole exon sequencing technology provides an opportunity to explore the suspected pathogenic genes of cholesterol stones.The purpose of this study was to screen out the suspected pathogenic genes of cholesterol gallstones in patients with genetic background by using WES technology,comparing with databases and filters and combining with bioinformatics analysis.In addition,the screening of core families,Sanger verification and preliminary verification of animal models provide new clues for the study of genetic pathogenesis of cholelithiasis.Saikosaponin D(SSD)is a monomer extracted from bupleurum chinensis.It has such pharmacological effects as anti-tumor,immunomodulation,anti-inflammatory,lipid-lowering and liver protection.This study is the first to use SSD in the field of cholesterol gallstone disease,to explore the effectiveness of SSD in preventing cholesterol gallstone and its possible protective mechanism.It also provides theoretical guidance for the development of new drugs for the treatment of cholesterol stones and the early prevention.Part 1:Whole exome sequencing to explore the pathogenic gene mutation and functional prediction of cholesterol gallstonesObjective: In this study,we used WES to explore the suspected pathogenic genes associated with the development of cholesterol gallstones in patients with genetic background.Methods: Complete data were collected from 21 patients with familial cholesterol gallstones and 5 healthy family members.About 5ml of peripheral venous blood was collected,genomic DNA was extracted,and WES was conducted.The output data was obtained through strict quality control system.Two screening strategies were adopted for data analysis:(1)Based on the priori lipid metabolism related genes and the previously reported pathogenicity variations,combined with literature retrieval,indepth data mining was conducted to obtain the suspected pathogenic mutation genes of cholesterol stones.(2)Based on the "Overlap" method of all diseased samples from eight families,the list of genetic variations shared by more than two families and the mutant genes that are common to more than two patients and more than two families,was obtained through the minimum mutation frequency filter and the online software SIFT,Mutation Assessor and Poly Phen2.The bioinformatics analysis of these mutant genes includes Gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The list of genes was submitted to the interactive gene retrieval tool(STRING)database,and a gene interaction network graph was obtained.Cytoscape version 3.4.0 software was used to visualize the network diagram of the interaction regulation of genes related to familial cholelithiasis,and the inserted molecular complex detection(MCODE)technology was applied to screen the module of the interaction network of mutant genes.The list of mutated genes was retrieved and the suspected pathogenic genes were obtained by combining the above bioinformatics analysis.Results: Based on a prior study,it was considered that lipid metabolism-related genes LRP2,LRP5,PLTP,OSBPL10,MUC5 B,ABCA1,ABCB11 and TGR5 may play an important role in the pathogenesis of cholesterol gallstone.Based on the screening principle of "Overlap" and bioinformatics analysis,it was found that PHLPP2,NOTCH1,SMARCA4,INTS1 and HUWE1,as well as ubiquitin-mediated protein hydrolysis pathway and protein digestion and absorption signal pathway may play a role in the occurrence and development of cholesterol gallstone.Conclusion: A large number of mutant genes were obtained through WES,and 13 disease-causing variants were finally located by combining screening strategy and bioinformatics analysis(based on prior LRP2,LRP5,PLTP,OSBPL10,MUC5 B,ABCA1,ABCB11 and TGR5).Based on "Overlap" screening,PHLPP2,NOTCH1,INTS1,SMARCA4 and HUWE1)as well as possible ubiquitin-mediated protein hydrolysis pathway and protein digestion and absorption signal pathway play an important role.These gene mutations and signaling pathways may play a role in cholesterol stone formation.Part 2:Whole exome sequencing and localization of pathogenic genes in a family of cholesterol gallstonesObjective: To explore a suspected pathogenic gene in a family with cholesterol stones,and to verify the accuracy of the results by comparing the suspected variant gene in the first chapter.Methods: Firstly,cholelithiasis screening was conducted in the family of cholesterol gallstones by collecting medical history data and abdominal ultrasound.Genomic DNA was extracted from peripheral venous blood of 6 members for WES,and data were output through the quality control system.Mutation screening was performed on sequencing data by comparing the database of 1000 Genomes Project,db SNP141 and EAS databases,excluding known mutations in the database and preserving mutations shared by patients but not normal people.Mutation Assessment,SIFT and Poly Phen2 were combined to predict the suspected pathogenic mutations,and the most likely pathogenic mutation genes were selected.Polymerase chain reaction(PCR)and Sanger methods were used to co-isolate and verify the candidate pathogenic loci in the family,and the results were retrieved from Pub Med and Google scholar.Results: Co-segregation of INTS1 gene variation and cholesterol stone disease was found in familial cholelithiasis.The gene variant is a new mutation.Conclusion: In this study,new INTS1 mutation of suspected pathogenic gene of family cholelithiasis was found.Part 3:Experimental study on the prevention of cholesterol gallstones by SSDObjective: The animal cholesterol stone model was used to verify the three-lipid metabolism related genes of ABCA1,ABCB11 and TGR5 m RNA based on the prior obtained in the first chapter.At the same time,the effects of SSD on the formation of dietary induced cholesterol gallstone in C57BL/6 mice were observed,the transcriptional levels of ABCA1,ABCB11 and TGR5 were detected,the effects of SSD on bile components were detected,and the effects of SSD on LKB1/AMPK signaling pathway were further explored.Methods: In animal experiments,the C57BL/6 cholesterol stone model was constructed by high-fat and high-cholesterol feeding method.40 males C57BL/6 mice,weighing 18-20 g,were bred adaptively for 2 weeks and divided into four groups by random number method(n=10),normal control group(group N-CON),conventional feed;model group(group LITH),diet high in fat and cholesterol.Ursodeoxycholic acid capsules group(group UDCA),high-fat and high-cholesterol feed diet and ursodeoxycholic acid capsule gavage treatment.Saikosaponin D group(group SSD),High fat and high cholesterol diet,at the same time SSD gavage treatment.The experiment period was 8 weeks,during which general conditions and weight changes of the animals were observed and recorded.After 8 weeks,the lithogenesis of each group was observed and the stone rate was recorded.The calculi were collected and qualitatively analyzed by using the potassium bromide tablet method.The lipid cholesterol,bile acid and phospholipids in bile were detected and the cholesterol saturation index(CSI)was calculated.Pathological HE staining was performed on liver tissues to analyze the liver cell injury and fat infiltration in each group.RNA was extracted from liver tissue,and the m RNA expression levels of cholesterol transporter ABCA1,bile acid transporter ABCB11 and bile acid receptor TGR5 were detected by RT-PCR.Total protein in liver tissue was extracted,and the activity levels of AMPK and LKB1 protein in liver cells were detected by Western blot to further explore the mechanism of SSD.Results: In week 8,LITH group was significantly heavier than N-CON group(P<0.05).The stone rate of LITH group was 100%,and the cholelith component was identified as cholesterol stone.The stone formation rate of UDCA group and SSD group was 20% and 30% respectively,which was significantly lower than that of LITH group(P<0.05).Bile cholesterol and phospholipids were significantly higher in the LITH group than in the n-con group(P<0.05),bile acid was significantly lower in LITH group than in NCON group;After treatment,the levels of cholesterol and phospholipids in UDCA group and SSD group were significantly lower than those in LITH group(P<0.05).The concentration of bile acid in UDCA group and SSD group was higher than that in LITH group,the difference was statistically significant(P<0.05).The CSI of the LITH group is greater than 1,indicating that cholesterol is saturated with bile formation,and the CSI of the UDCA group and the SSD group are both less than 1,indicating that both SSD and UDCA can alleviate the formation of supersaturated bile.In HE staining,no obvious abnormality was observed in N-CON group,presenting normal liver tissue status.In LITH group,a large number of fat vacuoles were observed.The fat vacuoles were full of hepatocyte cytoplasm,which could squeeze the nuclei to the edge.The nuclei were loose,and the ratio of nucleus-to-cytoplasm was significantly changed.Liver tissue damage was better in UDCA group and SSD group than in LITH group,and the pathological manifestations were between n-con group and LITH group.The results of RT-PCR showed that the relative expression levels of ABCA1 m RNA in NCON group,LITH group and SSD group were not significantly different(P>0.05).The expression of ABCB11 m RNA in LITH group was significantly lower than that in NCON group.The expression of TGR5 m RNA in LITH group was significantly higher than that in N-CON group.The relative expression of ABCB11 and TGR5 m RNA in the SSD group was significantly higher than that in the LITH group(P<0.05).Western blot results showed that SSD significantly increased the phosphorylation levels of LKB1 and AMPK in liver tissues(P<0.05).Conclusion: Compared with the N-CON group,the ABCA1 m RNA level in the LITH group did not change significantly,the ABCB11 m RNA level decreased significantly and the TGR5 m RNA increased significantly.SSD can change the lipid content of bile and alleviate cholesterol supersaturated bile production.SSD can prevent the formation of cholesterol stones by activating the LKB1-AMPK signaling pathway;ABCB11 and TGR5 may be the target of effect.