DNA Methyltransferase Inhibitor 5-AZA-DC Regulates TGFß1-Mediated Alteration of Neuroglial Cell Functions after Oxidative Stress.

5-AZA-DC is an efficient methylation inhibitor that inhibits methylation of target DNA. In this study, we explored the effects of 5-AZA-DC on the regulation of TGFß1 on target genes in neuroglial cell, as well as neuroglial cell functions under oxidative stress. The oxidative stress was constructed by editing CRISPR/Cas9 for knock out Ang-1 and ApoE4 genes. Cells were subjected to TGFß1OE (or shTGFß1) transfection and/or 5-AZA-DC intervention. Results showed that under oxidative stress, both TGFß1OE and shTGFß1 transfection raised DNMT1, but reduced TGFß1, PTEN, and TSC2 expressions in neuroglial cells. TGFß1 directly bind to the promoter of PTEN gene. 5-AZA-DC intervention lowered DNMT1 and raised TGFß1 expression, as well as promoted the binding between TGFß1 and promoter of PTEN. TGFß1OE caused a significant increase in the DNA demethylation level of PTEN promoter, while 5-AZA-DC intervention reduced the DNA demethylation level of PTEN promoter. Under oxidative stress, TGFß1OE (or shTGFß1) transfection inhibited neuroglial cell proliferation, migration, and invasion, promoted cell apoptosis. 5-AZA-DC intervention alleviated TGFß1OE (or shTGFß1) transfection caused neuroglial cell proliferation, migration, and invasion inhibition, as well as cell apoptosis. To conclude, these results suggest that 5-AZA-DC can be used as a potential drug for epigenetic therapy on oxidative stress damage in neuroglial cells. The findings of this research provide theoretical basis and research ideas for methylation drug intervention and TGFß1 gene as a possible precise target of glial oxidative stress diagnosis and treatment.

[Epigenetic regulation for spermatogenic dysfunction-related infertility in C57BL/6J male mice: An experimental study].

OBJECTIVE: To investigate the effect of epigenetic regulation on spermatogenic dysfunction-related infertility (SDI) in C57BL/6J male mice. METHODS: Sixty C57BL/6J male mice were randomly divided into a normal control and an SDI model group and the SDI model was established using the epididymis-targeting polypeptide CSA combined with indocyanine green-loaded free nanoparticles (ICG-NPS), busufan and dimethyl sulfoxide (DSMO). After intervention with 5-AZA-DC, the epididymides were collected from the mice for measurement of the rates of sperm DNA fragmentation (SDF), sperm acrosome integrity (SAI) and spontaneous acrosome reaction (SAR), amplification of the ERp29 gene by FISH, determination of the mRNA and protein expressions of DNMT1, ERp29, PTEN and TSC2 by quantitative real-time PCR and Western blot, and analysis of the ERp29, PTEN and TSC2 genes by methylated DNA immunoprecipitation sequencing (MeDIP-seq). RESULTS: After 5-AZA-DC intervention, statistically significant differences were observed between the normal control and the SDI model groups in the rates of SDF (ï¼»15.67 ± 1.33ï¼½% vs ï¼»30.15 ± 2.87ï¼½%, P < 0.05) and SAI (ï¼»65.33 ± 7.14ï¼½% vs ï¼»47.16 ± 3.45ï¼½%, P < 0.05), but not SAR (ï¼»11.52 ± 2.31ï¼½% vs ï¼»11.48 ± 2.27ï¼½%, P > 0.05). FISH confirmed evident amplification of the ERp29 gene in the SDI model but not in the normal control group. Compared with the baseline, the SDI model mice showed significant decreases after intervention in the mRNA and protein expressions of DNMT1 (ï¼»9.33 ± 1.15ï¼½ vs ï¼»7.01 ± 1.14ï¼½, P < 0.05; ï¼»15.66 ± 1.45ï¼½ vs ï¼»12.33 ± 1.27ï¼½, P < 0.05), but increases in those of ERp29 (ï¼»3.04 ± 1.13ï¼½ vs ï¼»6.54 ± 1.18ï¼½, P < 0.05; ï¼»4.37 ± 1.02ï¼½ vs ï¼»6.95 ± 1.03ï¼½, P < 0.05), PTEN (ï¼»3.25 ± 1.01ï¼½ vs ï¼»5.85 ± 1.04ï¼½, P < 0.05; ï¼»3.54 ± 1.01ï¼½ vs ï¼»5.17 ± 1.02ï¼½, P < 0.05) and TSC2 (ï¼»4.27 ± 1.16ï¼½ vs ï¼»6.98 ± 1.13ï¼½, P < 0.05; ï¼»3.83 ± 1.12ï¼½ vs ï¼»6.98 ± 1.13ï¼½, P < 0.05). No statistically significant differences, however, were found in the above parameters in the normal control group before and after intervention (P > 0.05). MeDIP-seq manifested 18 significantly differential genes were highly expressed and another 25 lowly expressed in the epididymal tissue of the model mice, all the former 18 down-regulated and all the latter 25 up-regulated after intervention, particularly ERp29, PTEN and TSC2. But there were no statistically significant differences in the expressions of the above genes in the control group (P > 0.05). MeDIP-seq also showed significant differences in the regional methylation levels of the Erp29, PTEN and TSC2 promoters in the epididymal tissue of the model mice (P < 0.05), but not in that of the normal controls after intervention (P > 0.05). CONCLUSIONS: A stable and efficient animal model provided valuable experimental evidence for the diagnosis and treatment of spermatogenic dysfunction-related infertility. ERp29 is an important gene involved in infertility and can be used as a potential target for epigenetic regulation in the treatment of infertility.

[Study on the effect of combined therapy of Chinese and Western medicines and gene therapy (GT) on cartilage end-plate calcification in rabbits with cervical intervertebral disc regression].

OBJECTIVE: To study the relationship between cartilage end-plate calcification (CEC) and cervical intervertebral discs regression (CIDR) in rabbits, and to study the inhibitory effect of combined therapy of Kanggu Zengsheng Capsule (KZC) ansforming growth factor-apsule (TGF-PLC) and igene therapy (GT) on CEC by measuring the thickness of CEC layer. METHODS: Thirty-five New Zeland rabbits of 4 months old were selected to establish cervical dynamic imbalance rabbit model for inducing CIDR (No disposal was given to rabbits in the normal control group). Seven months after operation, combined therapy of KZC and PLC were given, in doses calculated by body weight, to the modeled rabbits in the drug treated group with CEC of either superficial layer or full layer, twice a dantly by gavage for 30 successive days. While to those in the gene therapy group, the recombinant plasmid DNA with transforming growth factor-beta1 (TGF-beta1) was injected once their intervertebral discs (ID) of C(2-3) C(3-4) and C(4-5), 20 microl for each injection. One month later, all rabbits were sacrificed with periotic venous gas embolic method and their ID of C(4-5) (including partial body of the upper and lower vertebrae) was resected. The degree of CIDR was evaluated morphologically, and the thickness of CEC in rabbits was measured and compared between groups. RESULTS: Thickness of CEC in the model group, either of superficial layer or of full layer, was significantly more than that in the normal control group with significant difference. Both combined KZC and PLC therapy and gene therapy showed significant inhibitory effects on CEC in treating CIDR (P < 0.05). CONCLUSION: CEC is the initial factor of CIDR with highly positive correlation. Both combined therapy of KZC and PLC and gene therapy can significantly inhibit CEC.