| Object:(1) To study the effect of Chinese herbs of Jiedu-quyu-ziyin decoction on gene expression of DNA methylation regulation gene (DNMT1, Gadd45a), effect on gene expression of DNA methylation sensitive gene (CD70, IL-10, and PP2Ac), and effect on genomic DNA methylation level of CD4positive T lymphocytes of MRL/Lpr mice.(2) To study the relationship between the DNA methyaltion level of glucocorticoid receptor (GR) gene promoter1and serum concentration&mRNA expression level of GRa gene of peripheral blood white cells (PBWC) in patients with SLE.Methods:(1) Twenty-four six-week old MRL/Lpr mice were divided into two groups:twelve mice in model group and twelve mice in herb group. The model group and the herb group were given a gavage of distilled water and the same volume of Chinese herbs of Jiedu-quyu-ziyin decoction, respectively. Eight weeks later, all the mice were killed, and the samples of blood, urine, spleen, and lymph nodes were taken to store. Serum concentration dsDNA and C3were detected by enzyme-linked immunosorbent assay (ELSIA). Urine microalbumin and creatinine were also detected by ELSIA. Mice renal tissues were detected by hematoxylin-eosin (HE) staining and IgG immunofluorescence assay. Mice spleen CD4positive T lymphocytes were selected through immunomagnetic beads. Genomic DNA of CD4positive T lymphocytes were extracted by Genomic DNA Mini Preparation Kit with Spin Column. The global genomic DNA methylation levels of CD4positive T lymphocytes were detected through The MethylFlashTM Methylated DNA Quantification Kit (Fluorometric). The mRNA expression of DNA methylation regulation gene (DNMT1, Gadd45a) and DNA methylation sensitive gene (CD70, IL-10, and PP2Ac) were detected through real-time quantification PCR. (2) Collect ten patients with SLE, including2males, eight females, average age was25±8.5years, average course of disease was31±9.3months, and ten healthy controls,24±9.1years. The DNA methylation levels of glucocorticoid receptor (GR) gene promoter1of peripheral blood white cells (PBWC) in patients with SLE were detected through Bisulfite sequencing PCR (BSP). The mRNA expression of GRa and DNMT1of PBWC were detected through real-time quantification PCR. Serum concentration GRa and DNMT1were detected by enzyme-linked immunosorbent assay (ELSIA).Results:(1) Eight weeks later, urine ratio of microalbumin versus creatinine of herb group mice declined when compared with that of model group, there was statistic difference between them (p＜0.05). However, there were no statistic differences in serum dsDNA and C3between two groups (p＞0.05).(2) Eight weeks later, the global genomic DNA methylation level of MRL/Lpr mice CD4positive T lymphocytes of model group and herb group were3.108±2.261percent and8.008±5.304percent, respectively. There was statistic difference between them (p＜0.05).(3) Eight weeks later, the mRNA expressions of IL-10of MRL/Lpr mice CD4positive T lymphocytes of model group and herb group were3.8181±1.2687and1.0635±0.5114, respectively. There was statistic difference between them (p＜0.05). The mRNA expressions of PP2Ac in two groups were1.3408±0.6335and1.0404±0.4813, and those of CD70were0.9261±0.3325and1.3499±0.5841. There was no statistic difference between them, respectively.(4) Eight weeks later, the mRNA expressions of Gadd45a of MRL/Lpr mice CD4positive T lymphocytes of model group and herb group were2.2334±0.8857and1.0475±0.4132, respectively. There was statistic difference between them (p＜0.05). That of DNMT1was0.1647±0.0568and0.2813±0.0954, there was no statistic difference between them (p＞0.05).(5) The DNA methylation levels of glucocorticoid receptor (GR) gene promoter1of PBWC in patients with SLE and healthy controls were3.3±1.2percent and8.6±2.3percent, respectively. There was statistic difference between them (p<0.05),(6) The mRNA expression of GRa of PBWC in patients with SLE and healthy controls were16.511±11.885and10.242±6.413, respectively. There was statistic difference between them (p＜0.05). The mRNA expression of DNMT1of PBWC in patients with SLE and healthy controls were11.36±2.27and30.19±1.96, respectively. There was statistic difference between them (p＜0.05).(7) The serum concentration of GRa in patients with SLE and healthy controls were32.705±1.956and24.757±2.012, respectively. There was statistic difference between them (p<0.05). The serum concentration of DNMT1in patients with SLE and healthy controls were24.678±1.491and40.532±1.454, respectively. There was statistic difference between them (p＜0.05).Conclusions:(1) Through suppression of mRNA expression of DNA methylation regulation gene Gadd45a, the Chinese herbs of Jiedu-quyu-ziyin decoction can improve global genomic DNA methylation level of CD4positive T lymphocytes of MRL/Lpr mice, and inhibit mRNA expression of DNA methylation sensitive gene (IL-10and PP2Ac).(2) The serum concentration of DNMT1and the mRNA expression of DNMT1of PBWC in patients with SLE were increased, and the DNA methylation levels of GR gene promoter1of PBWC in patients with SLE were increased, those inducing the decline of serum concentration and mRNA expression of GRa of PBWC in patients with SLE. |
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