RESUMO
Objective To observe the activation of rat hepatic stellate cells (HSC-T6) and the changes of methyltransferase (DNMT)1,DNMT3a mRNA expression with different doses of sodium arsenite stimulation.Methods HSC-T6 cells were exposed to a final concentration of 0 (control),5 (low dose),15 (medium dose) and 25 (high dose) μmol/L sodium arsenite in culture medium for 24,48 and 72 h,cells and cell culture supernatant were harvested.Enzyme-linked immunosorbent assay (ELISA) kit was used to measure fibrosis factors contents of type Ⅰ collagen (COL-1),type 11Ⅲ collagen (COL-3) and alpha smooth muscle actin (α-SMA) and quantitative real-time PCR was used to detect the expression levels of DNMT1 and DNMT3a mRNA.Results Different arsenic exposure time (24,48,72 h) had a significant effect on COL-1,COL-3 and α-SMA contents in HSC-T6 cells (F =249.574,328.493,3 157.436,all P ＜ 0.01);Different arsenic exposure content (low,medium,high dose groups) had a significant effect on COL-1,COL-3 and α-SMA contents in HSC-T6 cells (F =3 946.521,1 006.399,13 025.770,all P ＜ 0.01).After arsenic exposure for 24 and 48 h,the expression levels of DNMT1 mRNA in high dose group (4.33 ± 0.24,2.34 ± 0.43) were higher than those of control group (1.00 ± 0.00,1.00 ± 0.00,all P ＜ 0.05).At the same arsenic exposure levels (low,medium or high dose),the expression level of DNMT1 mRNA was declined with prolongation of sodium arsenite stimulation time (all P ＜ 0.05).After arsenic exposure for 48 and 72 h,the expression levels of DNMT3a mRNA in high dose group (2.23 ± 0.50,5.02 ± 0.23) were higher than those of control group (1.00 ± 0.00,1.00 ± 0.00,all P ＜ 0.05).The expression levels of DNMT3a mRNA in medium and high dose groups at 72 h (3.80 ± 0.14,5.02 ± 0.23) were higher than those of 24 h (3.03 ± 0.12,0.42 ± 0.15,all P ＜ 0.05).Conclusion HSC-T6 cells are obviously activated with pro-fibrotic effect;the expression levels of DNMT1 and DNMT3a mRNA are both up regulated in HSC-T6 cells after being exposed to sodium arsenite.
RESUMO
Objective To study the mechanism of liver fibrosis in rats caused by chronic exposure through drinking water containing sodium arsenite,to identify the differential proteins via proteomics technique.Methods Totally 40 healthy 8-week-old male Sprague Dawley (SD) rats of specific pathogen free (SPF) grade were randomly divided into 4 groups,which were control group (deionized water),0.68,1.36 and 2.73 mg/kg sodium arsenite (iAs3+) treated groups,respectively.The rats were fed with iAs-treated drinking water freely for 24 consecutive weeks.Twenty-four hour urine sample,blood and liver samples were collected.Hepatic fibrosis indices,specifically,type Ⅲ precollagen (PC Ⅲ),type Ⅳ collagen (Ⅳ-C),hyaluronic acid (HA) and laminin (LN) were detected by enzymelinked immunoassay (ELISA).Based on the isobaric tags for relative and absolute quantitation (iTRAQ) reagent 8-plex experiment,combined with 2DLC-MS/MS,the proteins in rats liver tissue of the medium dose group and the high dose group were compared with the those of control groups.Results ①The serum HA contents in the C (control) group,the L (low dose) group,the M (medium dose) group and the H (high dose) group were (198.51 ± 16.64),(218.39 ± 34.98),(261.72 ± 30.56) and (297.31 ± 35.72) ng/L;the serum PCⅢ contents in C,L,M and H groups were (15.32 ± 2.15),(16.78 ± 2.64),(19.51 ± 0.85) and (21.42 ± 1.63) μg/L;the serum LN contents in C,L,M and H groups were (734.57 ± 86.00),(792.65 ± 94.15),(916.83 ± 84.40) and (1 008.09 ± 64.17) μg/L;the serum Ⅳ-C contents in C,L,M and H groups were (52.34 ± 14.65),(59.72 ± 12.84),(74.38 ± 4.83) and (78.46 ± 4.30) μ.g/L,respectively.The differences in serological indices of liver fibrosis between-groups were statistically significant (F =21.136,19.957,22.007,14.288,all P ＜ 0.05).In multiple comparison of serum HA,PCⅢ and LN,there were no statistical significant differences between L group and C group.M and H groups were higher than L group and C group,significant statistical difference was found between H group and M group (all P ＜ 0.05).②Combining iTRAQ with 2DLC-MS/MS,based on the confidence threshold of protein (unused protScore) ＞ 1.3 and at least 1 matched peptides within the 95％ confidence interval,2 948 proteins were identified.Totally 2 162 proteins were detected in three groups compared with Venn diagram,after removing significant different proteins in C group,687 up-regulated proteins and 548 down-regulated proteins were identified in M group;633 up-regulated proteins and 519 downregulated were found in H group;the differences of protein expression between M and H groups were not statistically significant (P ＞ 0.05).③Up-regulated proteins related to the metabolism including AS3MT,MAT,SHMT,CHDH,CTH,CSAD and BHMT in M and H groups;of the two kinds of proteins of MTR,METK1 was up-regulated and F1LRB8 was down-regulated.Proteins associated with GSH including Gsta1,Gsta4,Gsta5,Gstt1,Gstt2,Gstk1,Gstp1,Gstm1,Gstm2,Gstm3,Gss,Gpx1,Gpx4,Esd,Hagh,Glo1,Mgst1 and B6DYQ5 which were all up-regulated.Proteins associated with liver fibrosis were Hic-5,Gss and six kinds of Tpm,and six kinds of Tpm subunits including two kinds of Tpm1,three kinds of Tpm2 and one kind of Tpm3 which were all up-regulated.Conclusions There is liver accumulation of arsenic after chronic arsenic exposure and resulting in liver fibrosis and decline of liver function.Expressions of AS3MT,MTR,MAT,SHMT,BHMT,CHDH,CTH and CSAD are up-regulated;arsenic meta bolism methionine cycle,folic acid cycle and sulfur transfer pathways are closely related.GSH plays an important role in arsenic metabolism and liver fibrosis,Hic-5,GSS and TPM may be associated with the occurrence of liver fibrosis.