OBJECTIVETo put the insight into the trichloroethylene (TCE)-induced effect on the differential expression of subcellular proteins in human normal liver cell line (L-02).

METHODSThe membrane proteins and nuclear proteins of TCE-treated (8.0 mmol/L) group and controls were extracted by subcellular proteome extraction kit, respectively. The TCE-induced differentially expressions were analyzed by a two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight spectrometry (MALDI-TOF-MS). Bioinformatics analysis was used to reveal the biological processes and predict transmembrane domains of differential expressed proteins. The expression of ATP synthase subunit beta (ATP5B), heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) and far up steam element-binding protein 1 (FUBP1) were measured under TCE treatment by Western blot.

RESULTSAfter TCE treatment for 24 h in L-02 cells, 14 membrane proteins and 18 nuclear proteins were identified as differential expression. After treated with TCE in concentrations of 0, 2.0, 4.0 and 8.0 mmol/L for 24 h, the relative levels of ATP5B expression were 1.00±0.03, 1.21±0.14, 1.25±0.12 and 1.48±0.17 (F = 8.51, P = 0.007), the relative levels of hnRNP H2 expression were 1.00±0.09, 1.22±0.15, 1.43±0.21, 1.53±0.17 (F = 6.57, P = 0.015), respectively; the relative levels of FUBP1 expression were 1.00±0.11, 0.91±0.07, 0.73±0.04 and 0.67±0.03 (F = 15.81, P = 0.001), respectively, which were consistent with the results in proteomics. The bioinformatics analysis showed that the most dominant biological process were involved in RNA processing (10 proteins, P = 2.46×10(-6)), especially in RNA splicing (9 proteins, P = 1.77×10(-7)).

CONCLUSIONThe exposure of TCE could alter the expression of membrane proteins and nuclear proteins in L-02 cells. These abnormal expressed proteins involved in RNA splicing would provide novel clues for further understanding of TCE-induced hepatotoxicity.

Blotting, Western ; Cell Line ; DNA Helicases ; DNA-Binding Proteins ; Hepatocytes ; Heterogeneous-Nuclear Ribonucleoprotein Group F-H ; Humans ; Mitochondrial Proton-Translocating ATPases ; Proteome ; Proteomics ; RNA Processing, Post-Transcriptional ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Trichloroethylene

Objective:To investigate the relationship between single nucleotide polymorphic (SNP) loci of PLCE1 gene and primary nephrotic syndrome (PNS) and its response to glucocorticoid therapy in Guangxi Zhuang children. Methods:It was a retrospective cohort study. One hundred and fifty-five Guangxi Zhuang children with PNS in the Affiliated Hospital of Youjiang Ethnic Medical College from October 2020 to May 2022, and 100 healthy Zhuang children during the same period as controls were included. Four SNP loci including rs17109674, rs10786156, rs3740360 and rs2274224 of PLCE1 gene were selected and high-throughput sequencing was used to analyze the genotypes. Logistic regression analysis model was used to analyze the correlation between each SNP locus and onset of PNS and steroid-resistant nephrotic syndrome. The SHEsis online software was used to analyze the link disequilibrium of each SNP locus, and construct the haploid type. Results:(1) Logistic regression analysis results showed that AC+CC genotype (AA as reference, OR=0.449, 95% CI 0.256-0.786, P=0.005), AC genotype (AA as reference, OR=0.354, 95% CI 0.188-0.667, P=0.001) and C allele gene (A as reference, OR=0.615, 95% CI 0.390-0.971, P=0.037) of rs3740360 were correlated with the risk of PNS in children. The genotypes and allele genes of rs17109674, rs10786156, rs3740360 and rs2274224 were not associated with the risk of steroid-resistant nephrotic syndrome in children (all P>0.05). (2) Strong linkage disequilibrium existed between rs10786156 and rs2274224 ( D'=0.702, r2=0.484). rs17109674 and rs10786156 ( D'=0.128, r2=0.007), rs17109674 and rs3740360 ( D'=0.142, r2=0.007), rs17109674 and rs2274224 ( D'=0.045, r2=0.001), rs10786156 and rs3740360 ( D'=0.255, r2=0.023), and rs3740360 and rs2274224 ( D'=0.281, r2=0.028) all had weak linkage disequilibrium. (3) The haploid AGCG ( OR=0.282, 95% CI 0.079-1.008, P=0.038), GGCC ( OR=0.327, 95% CI 0.111-0.967, P=0.034) and GGAG ( OR=4.616, 95% CI 1.179-18.069, P=0.016) were all correlated with the risk of PNS in children. Conclusions:AC genotype, AC+CC genotype, and C allele gene of rs3740360 SNP locus may reduce the risk of PNS in Guangxi Zhuang children. Haploid AGCG and GGCC may be associated with decreased incidence of PNS, while GGAG may be associated with increased incidence of PNS in Guangxi Zhuang children. The genotypes and alleles of 4 SNP loci are not associated with the risk of steroid-resistant nephrotic syndrome.

OBJECTIVETo compare the DNA methylation-related alteration induced by trichloroethylene (TCE) in human hepatic L-02 cells (L-02 cells) and SET deficient cells, and reveal the role of SET on the mechanisms in TCE-induced epigenetic pathway.

METHODSThe L-02 cells and pre-established SET deficient cells were treated with different TCE concentrations, and the changes of total cell viability, DNA methylation level and DNA methyltransferases (DNMTs) activity were measured, respectively. In addition, the TCE-induced alteration in the protein expression of DNMT1, DNMT3a and DNMT3b were analyzed by Western blotting.

RESULTSAfter treatment with TCE for 24 h, the cell proliferation level was significantly decreased in both cell lines. When concentrations of TCE were 0, 1.0, 2.0, 4.0 and 8.0 mmol/L, the proliferation levels of L-02 cells were 100.00±2.70, 83.34±2.38, 75.56±4.51, 71.67±2.77 and 66.67±1.63, respectively (F = 58.29, P < 0.001); the cell proliferation levels of SET deficient cells were 101.12±1.67, 85.01±2.33, 79.44±1.67, 78.337±3.89 and 76.11±3.33, respectively (F = 42.41, P < 0.001). When concentration of TCE reached 4.0 mmol/L, the difference of cell proliferation level between two groups was statistically significant (t = -3.51; P = 0.013). After treated by TCE for 24 h, the global DNA methylation significantly decreased in both cell lines (F value was 212.87 and 79.32, respectively, P < 0.001). The difference between two groups was not statistically significant. After treated by TCE for 24 h, the methyltransferases activities were significantly decreased in both cell cells (F values were 77.92 and 113.80, respectively, P-0.001). The SET deficiency could inhibit the decrease of methyltransferases activity under TCE treatment. When the concentration of TCE reached 8.0 mmol/L, the enzymatic activity of L-02 cells and SET deficient cells decreased to 67.61%±2.85% and 72.97%± 1.94%, respectively. The difference between two groups was statistically significant (t = -3.94, P = 0.008). After treated with TCE for 24 h, concentrations of TCE were 0, 1.0, 2.0, 4.0 and 8.0 mmol/L, and the relative protein levels of DNMT1 in normal L-02 cells increased significantly to 1.00±0.03, 1.28±0.04, 1.20±0.04, 1.62±0.05, 1.43±0.04 (F = 103.00, P < 0.001); In SET deficient cells, the expressions of DNMT1 were 1.00±0.04, 0.96±0.02, 1.19±0.05, 0.85±0.03, 0.83±0.03, which was significantly down-regulated under TCE treatment (F = 44.18, P < 0.001).

CONCLUSIONSET deficiency can significantly attenuate the TCE-induced decreases of cell viability and DNMTs activity, as well as alteration of protein expression of DNMT1 in L-02 cells, which indicated that SET was involved in the mechanism of TCE-induced cytotoxicity and epigenetic pathway in L-02 cells.

Cell Line ; Cell Survival ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; DNA Methylation ; Humans ; Liver ; Trichloroethylene

Based on the modern anatomy and physiology, the referred pain of myofascial trigger points of each muscle is integrated; compared with the twelve meridians as well as conception vessel and governor vessel, the similarity of their position and running course is observed. With the current research progress of myofascial trigger points and fasciology, based on the running course of referred pain of trigger points, combined with fascia mechanics, nerve and vascular, the location of acupoints and meridians, as well as the relationship between acupoints and meridians, are discussed.
Acupuncture Points ; Humans ; Meridians ; Muscles ; Pain, Referred ; Trigger Points