| Objective: In this study,we investigated the changes in gene expressions of lipid metabolism-related genes in hepatic tissue of patients with hereditary cholesterol gallstone(HCGD)of Chinese Han nationality.On the other hand,in our study,whole exome sequencing(WES)was used to screen pathogenic candidate genes by analyzing genomic DNA samples from a HCGD pedigree of Chinese Han nationality family.To identified novel mutation gene or loci of HCGD by using bioinformatics analyze the suspected gene.At the same time,the present study was aimed to investigate whether the Yinchenhao Decoction(YCHD)could prevent the formation of lithogenic diet(LD)-induced cholesterol gallstones in C57BL/6J mice and attempted to elucidate potential mechanisms.Methods: Part1:The blood samples and surgical specimens of hepatic tissues from 9 patients with HCGD(HCGD group),9 patients with sporadic cholesterol gallstone(SCGD,SCGD group),and 7 hepatic hemangioma patients without gallstone(control group)were collected.the serum levels of total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL),low-density lipoprotein(LDL),apolipoprotein A1(Apo A1),apolipoprotein B(Apo B),lipoprotein a(Lp(a))as well as total bile acid(TBA)were determined by enzymatic assay.The hepatic tissue m RNA expression of 7 NRs in liver tissues,which included liver X receptor(LXRα),farnesoid X receptor(FXR),sterol regulatory element binding protein 2(SREBP2),estrogen receptors ɑ and β(ERα/β),G protein-coupled receptor 30(GPR30),peroxisome proliferator activated receptor γ(PPARγ)were examined by q RT-PCR.The hepatic tissue m RNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR),cholesterol 7α-hydroxylase(CYP7A1),cholesterol 12α-hydroxylase(CYP8B1),adenosine triphosphate binding cassette subfamily B member 4(ABCB4),adenosine triphosphate binding cassette subfamily B member 11(ABCB11),apical sodium dependent bile acid transporter(ASBT),organic solute transporter α(OST α),organic solute transporter β(OST β),lipid binding protein(ILBP),adenosine triphosphate binding cassette subfamily G member 5(ABCG5),adenosine triphosphate binding cassette subfamily G member 8(ABCG8)and Niemann Pick C1 like 1(NPC1L1)were examined by q RTPCR.Part 2: We studied a 4-generation family(including 18 members)with five members affected with CGD.The proband(III2)and four family members(II3,II4,II6,III2)of a non-consanguineous Chinese family were enrolled for analysis.Genomic DNA(g DNA)was extracted from peripheral blood leukocytes obtained from family members with CGD(II3,II4,II6,III2)and 1 normal control(III4)of this HCGD pedigree.Illumina Hiseq 2000 WES was performed,and the identified SNVs and INDELs variants were filtered to exclude known polymorphisms(variants listed in 1000 Genomes and inhouse databases)with a minor allele frequency(MAF)>0.05).Furthermore,the variants with normal control individuals(III3)of the family were filtered to screen candidate mutation.The suspected variants were predicted by SIFT,Poly Phen,Radial SVM,FATHMM and LR software for further pathogenicity predication and analysis.Part 3: CGD formation was induced in C57BL/6J mice were fed on a lithogenic diet(LD)for 8 weeks.After acclimation for 2 weeks,40 healthy male C57BL/6J mice were randomized into the following 4 experimental groups: i)control group(NG),ii)lithogenic diet(LD),iii)lithogenic diet plus ursodeoxycholic acid(LD+UDCA),iiii)lithogenic diet plus yinchenhao decoction(LD+YCHD).The mice were acclimated for 2 weeks,fed the LD for 8 weeks,and then treated with the LD and the simultaneous oral administration of either UDCA or YCHD for 8 weeks.After 8-week treatments,the blood samples were obtained from the orbital venous.The liver tissues were excised immediately for histological investigation and q RT-PCR detection.Bile samples were obtained from the dissected gallbladders,stored at-20 °C,and diluted with distilled water prior to analysis.Infrared spectroscopy(IRS)was used in qualitative analysis of gallstones.The gallstone formation and morphology of liver and gallbladder were observed by stereoscopic microscopy.HE staining was performed for observing pathological changes of hepatic tissues.Intracellular lipid droplets were visualized using Oil Red O staining.The serum levels of total TC、TG、HDL、LDL as well as TBA were determined by enzymatic assay.The levels of bile cholesterol(Ch),phospholipids(PLs),and bile acids(BAs)using automatic biochemical analyzer.The cholesterol saturation index(CSI)was calculated according to Carey’s critical tables.The hepatic tissue m RNA expression of NRs in liver tissues,which included Srebp2、Erɑ、Erβ and Gpr30 were examined by q RT-PCR.Results: Part 1: The serum level of TBA in HCGD group was significantly reduced compared with control group and SCGD group(both p<0.05),while no significantly differences were noted in other lipid metabolism related indicators among the three groups(all p>0.05).(1)Compared with control group,the m RNA expression levels of ERα/β,SREBP2,PPARγ and NPC1L1 m RNA were significantly increased in HCGD group,and the FXR,OSTα as well as ILBP m RNA expression levels were significantly increased in SCGD group(all P<0.05),CYP7A1 m RNA expression level was significantly decreased in SCGD group(P<0.05);(2)the m RNA expression levels of LXRα,GPR30,CYP8B1,HMGCR,ABCB4,ABCB11,ASBT,OSTβ,ABCG5,ABCG8 showed no significant differences among the three groups(both P>0.05);(3)in HCGD group,the m RNA expressions of ERα and ERβ were negatively correlated with the serum TBA level(r=-1.000,P= 0.000;r=-0.989,P= 0.011).Part 2: There are five affected individuals in the four-generation Chinese family.By filtered variants and bioinformatics analysis,we revealed that 5 mutation genes in four affected individuals(II3,II4,II6 and III2),but were not found in control individuals(III3).However,no SNVs variants were found in four patients with CGD.In total,5 SNVs were present in the proband(III2)and her aunt(Ⅱ: 6): ESPNP(n.1440C>G),PI4KA(c.6083+184 A>G),MUC16(c.36796-24C>T;c.37698C>T: p.Ser12566 Ser;c.40203C>T: p.Pro13401 Pro).4 SNVs were present in the proband(III2)and her mother(Ⅱ: 4): SORCS2(c.1072-32C>T,c.3311+913C>T),MUC16(c.20694T>C: p.His6898His;c.36067 +50C>A).1 SNVs were present in the proband(III2),her father(Ⅱ: 3)and her aunt(Ⅱ: 6): MUC16(c.41946-168A>G).Two missense mutations were present in the her father(Ⅱ: 3)and her aunt(Ⅱ: 6): BAGE3(c.14T>C: p.Val5Ala).We identified a novel heterozygous nonsense variant,c.38224A>T in MUC16,that resulted in a p.Thr 12742 Ser substitution.This mutation was predicted to be pathogenic by SIFT(SIFT=0).A heterozygous nonsense variant(c.40204A>C: p.Lys13402Gln)of MUC16 gene was predicted to be damaged by Poly Phen2(Poly Phen2 = 0.974).Whether MUC16 and SORCS2 are candidate genes of HCGD remains need to be further verified.Through functional analysis,we speculate that MUC16 more likely to be candidate gene.Part 3: In our study,the NG group had a gallstone formation rate of 0%(0/10);the gallstone formation rate in the UDCA group and YCHD group were 30%(3/10)and 40%(4/10),respectively.All gallstones were qualitative diagnosed cholesterol gallstone by IRS.(1)The body weights of the mice,the serum levels of TG and TC,the bile levels of Ch,PLs and CSI in LD group were significantly increased compared with NG group(all p<0.01);while the serum levels of HDL-C and TBA,the bile level of BAs were significantly decreased compared with NG group(all p<0.01);no significantly differences were noted in the serum levels of LDL-C between the two groups(p>0.05).(2)The body weights of the mice,the serum levels of TG,the bile levels of CSI in UDCA group and YCHD group were significantly decreased compared with LD group(all p<0.01);the serum levels of TBA,the bile levels of PLs and BAs in UDCA group and YCHD group were significantly increased compared with LD group(all p<0.05);the serum level of TC in YCHD group was significantly decreased compared with LD group(all p<0.05);(3)the serum levels of TC and LDL-C in UDCA group,the serum levels of HDL-C and LDL-C in YCHD group were no significantly differences compared with LD group(all p>0.05).HE and Oil Red O staining shown severe steatosis and lobular inflammation in the livers of the LD group.Compared with LD group,the extent of hepatocellular steatosis and inflammation were markedly decreased in UCDA group and YCHD group.(1)Compared with NG group,the m RNA expression levels of Erα and Hmgcr m RNA were significantly increased in LD group(all P<0.01),and the Gpr30 m RNA expression level was significantly decreased in LD group(P<0.05),the m RNA expression levels of Erβ m RNA showed no significant differences between groups(P>0.05).(2)Compared with LD group,the Gpr30 m RNA expression level in UDCA group and the Erβ m RNA expression level in YCHD group were significantly increased(P<0.01).However,the Srebp2 and Erα m RNA expression level in YCHD group were significantly decreased(all P<0.05).The m RNA expression levels of Srebp2,Erα,Erβ and Hmgcr in UDCA group,Gpr30 and Hmgcr in YCHD group showed no significant differences compared with LD group(both P>0.05);(3)in YCHD group,the m RNA expressions of ERα and ERβ were negatively correlated with the serum TBA level(r=-1.000,P= 0.000;r=-0.989,P= 0.011).Conclusions:(1)The genes expressions of ERα/β,SREBP2,PPARγ and NPC1L1 are up-regulated in the liver tissue of HCGD patients,which may play important roles in the pathogenesis of HCGD.(2)MUC16 and SORCS2 are probably novel candidategenes for HCGD.The c.38224A> T mutation of the MUC16 gene probably underlies the HCGD in this family.Our study expands the spectrums of MUC16 mutations and contributes to genetics counselling of families with HCGD.(3)YCHD inhibited the formation of gallstone;its effects were mediated by ERα and SREBP2 m RNA downregulated,which increase the efflux of bile salt.Furthermore,these effects upregulate ABCG5/G8,which decreased the bile Ch and CSI,increased the bile BAs,ultimately leading to the suppression the LD-induced cholesterol gallstone formation. |
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