[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.

[Regulatory Effect of Zishen Yutai Pill on the Biological Behavior of Oocytes in Immune Infertility Model Rats].

Objective To observe the intervention effect of Zishen Yutai Pill (ZYP) on the oo- cytes of infertile rats with positive anti-sperm antibody (AsAb). Methods AsAb positive immune infertil- ity model was established using active immunity. Totally 100 female SD rats were randomly divided into the blank control group, the model group, low, middle, and high dose ZYP groups, 20 in each group. Dif- ferent medications were administered to rats in corresponding groups by gastrogavage from the 22nd day of modeling to the 36th day, once per day. Normal saline (5 mL/kg) was administered to rats in the model group by gastrogavage. ZYP at 0. 075, 0.100, 0.150 g/mL was respectively administered to rats in low, middle, and high dose ZYP groups. AsAb and transforming growth factor ß (TGF-ß) levels were meas- ured by ELISA. Oocytes were collected after rats were sacrificed. mRNA and protein expression levels were detected using Real-time quantitative polymerase chain reaction ( RT-PCR) and Western blot. Oo- cyte apoptosis rate was determined by flow cytometry (FCM) and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). The oocyte proliferation level was detected by cell count- ing kit 8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU). Oodyte migration and invasion was determined u- sing invasion chamber (Transwell) and scratch experiment. Results Compared with the blank control group, the apoptosis of oocytes increased, the proliferation of oocytes was reduced, the capabilities of migration and invasion were enhanced, mRNA and protein expression levels of TGF-ß were significantly down-regulated (P <0. 05). Compared with the model group, the apoptosis of oocytes was reduced, the proliferation of oocytes was increased, the capabilities of migration and invasion were enhanced, mRNA and protein expression levels of TGF-ß were significantly up-regulated in the high dose ZYP group (P < 0. 05). Besides, their effects were obviously superior in the high dose ZYP group than in low and middle dose ZYP groups (P <0. 05). Conclusion The application of high dose ZYP could obviously up-regulate the level of TGF-ß and improve the function of oocytes.

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.

Establishment of composite experimental animal model of brain edema after cerebral hemorrhagic.

BACKGROUND: The aim of our study was to explore the establishment of an animal model of compound cerebral edema and assist the study of brain edema. METHODS: Sprague Dawley (SD) rats were randomly divided into 6 groups: Group A (IV collagenase + heparin autologous blood + non-heparin autologous blood), Group B (heparin autologous blood), Group C (IV collagenase), Group D (non-heparin autologous blood), Group E (control), Group F (sham); groups A, B, C, and D were modeled, group E received no treatment, and group F was injected with normal saline. The Longa/Bederson scales were used to test limb symmetry and score neurological deficits. The composition of brain tissue were measured by potassium (K+), sodium (Na+), and calcium (Ca2+). The glial cells were also subjected to primary culture and identification. The flow cytometry technique (FCM) and terminal uridine nick-end labeling (TUNEL) test were used to detect for the apoptosis of glial cells. Cell counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to observe the proliferation of glial cells; quantitative real-time polymerase chain reaction (qPCR) and western blot measured glial cell matrix metalloproteinase 9 (MMP-9) hole channel protein 4 messenger (m)RNA, and protein expression. The scratch and transwell tests were used to study the glial cell migration and invasion. RESULTS: Among all groups, according to the results of the Longa/Bederson scores of neurologic impairment, brain tissue water content, and electrolyte K+, Na+, Ca2+, group A showed the most significance (P<0.05). Group A showed the highest rate of apoptosis (32.27±3.33), least proliferation (5.33±1.14), and migration and invasion ability were the weakest (85.35±8.11; 8.08±1.96). The mRNA (9.31±0.84; 15.69±1.77) and protein (4.25±0.27; 4.34±0.33) of MMP-9 and aquaporin-4 (AQP4) were the most significant in group A (P<0.05). Inter-group comparison of groups B, C, and D and between groups E and F yielded no significant differences (P>0.05). CONCLUSIONS: By using the obvious signs of brain edema, we were able to establish a composite experimental animal model for the study of brain edema after cerebral hemorrhage, to provide a reliable experimental basis for the study of brain edema.

[Preliminary experimental study on the influence of different tidal volume and frequency of normal minute ventilation on the dynamic changes of carotid blood oxygen in living goats].

Objective: On the basis of preliminarily verifying the use of ultra-fast reaction polymer matrix optical fiber oxygen sensor and its measuring system to record the continuous and dynamic changes of carotid artery oxygen partial pressure (PaO2), in order to analyze and discuss the influence of lung ventilation on the continuous and dynamic changes of PaO2, we designed a whole animal experimental study in vivo. Methods: Four hybrid goats were selected, and the skin was cut and exposed directly under general anesthesia and tracheal intubation. The oxygen sensor, connected with the measuring system, was inserted directly into the left carotid artery to continuously record the dynamic changes of PaO2. With normal minute ventilation,mechanical ventilation is implemented through three tidal volumes: normal tidal volume (VT=15 ml/kg, Rf=20 bpm), half tidal volume (halved VT, doubled Rf) and double tidal volume (doubled VT, halved Rf). Each tidal volume was stable for 10~15 min respectively. We analyzed and calculated the average values of PaO2, the fluctuation magnitudes of PaO2 changes between breaths of last 180 s and the delay times of lung-carotid artery were. We analyzed the effects of different tidal volumes. Results: The heart rate and blood pressure of living goats were maintained stable during the mechanical ventilation experiment with normal ventilation volume Lung-carotid artery delay time is 1.4~1.8 s (about 3 heartbeats at this time). Under normal tidal volume of mechanical ventilation, the average value of PaO2 was (102.94±2.40, 99.38~106.16) mmHg, and the fluctuation range was (21.43±1.65, 19.21~23.59) mmHg, accounting for (20.80± 1.34, 18.65~22.22)% of the average value. Under the condition of halving tidal volume, the average value of PaO2 was maintained at (101.01±4.25, 94.09~105.66) mmHg, which was slightly decreased but not significant (P>0.05 compared with normal mechanical ventilation), but the fluctuation range of PaO2 was significantly reduced to (18.14±1.43, 16.46~20.05) mmHg, accounting for 17.5% of the average value. Under double tidal volume mechanical ventilation, although the average value of PaO2 increased slightly remained at (106.42±4.74, 101.19~114.08) mmHg (P>0.05 compared with normal mechanical ventilation and P<0.05 compared with half tidal volume mechanical ventilation), the fluctuation magnitude of PaO2 increased significantly to (26.58±1.88, 23.46~28.46)mmHg. Conclusion: Inspiration and expiration of normal lung ventilation are the initial factors for the increase and decrease of PaO2 in carotid artery. Under normal ventilation, halving tidal volume and doubling tidal volume significantly changed the fluctuation magnitude of PaO2, but the average value of PaO2 changed only slightly, while the lung-carotid delay time was similar.

[Ultra-fast response polymer optical fiber oxygen measurement device and its preliminary experimental report on continuous dynamic change of arterial oxygen partial pressure under mechanical ventilation in living animals].

Objective: We tried to implant the ultra-fast polymer optical fiber chemical oxygen sensor （POFCOS） into arterial blood vessel,connect with photoelectric conversion measurement system to record the continuous dynamic rapid changes of arterial PO2(PaO2) in whole living animals. It should be the experimental evidence for the new theory of holistic integrative physiology and medicine(HIPM) forexplain the mechanism of respiratory control and regulation in whole circusof respiration-circulation-metabolism. Methods: ①Fabrication of ultrafast POFCOS, calibration and its measuring system: The distal part of 2 m optical fiber was heated and pulled until it became a tapered tip. After cleaning and drying, the tip of 1 mm tapered optical fiber was dip-coated into the luminophore doped polymer solution, then was slowly pumped out while solvent was quickly evaporated to form an oxygen sensing tip, which was dried at room temperature for 24 hours. ②Animal experiments: Under general anesthesia and intubation, goatwas mechanically ventilated with 40%~60% oxygen. We exposed both right and left carotid arteries and the left femoral artery by skin cutting, and inserted the POFCOS directly into the arteries via indwelling catheter. The end of POFCOS were connected to the personal computer through optical fiber, excitation and detection Y-type optical fiber coupler through photoelectric conversion, so as we can realize the continuous dynamic response of living goat carotid PaO2 under mechanical ventilation. We mainly analyzed the intra-breath wave-form alternate increase and decrease of PaO2 and their time delay between lung and carotid arteries.We completes breathing control whole loop to explain the mechanism of mutual breathing and the switching of inspiration and exhalation. Results: The POFCOS has a very fast T90 response time was set 100 ms for liquid. When the heart rate of 40%~60% oxygen mechanical ventilated living goat was ~110 bpm, the PaO2 of left and right carotid artery showed a same wave-sizeup and down following with the inspiration and expiration of ventilator, with a range of up to 15 mmHg. There weresignificant noises of PaO2 change recorded in the left femoral artery. The lung-carotid artery time delay is 1.5~1.7 s after inhalation and exhalation, PaO2 at both left and right carotid arteries starts toincrease and decrease. After two-three heartbeats after the start of lung ventilation, thealternate up-down wave-form information of the arterialized pulmonary vein blood after pulmonary capillaries waspumpedby left ventricle to the position of peripheral chemoreceptors,thus realizing the whole cycle of inhalation and exhalation. It alternately interrupted inhalation, i.e. switching inhalation to exhalation, and then interrupted exhalation,i.e. switching exhalation to inhalation. Conclusion: The ultra-fast reactive implantableoxygen sensor and its measuring system can measure the physiological waveform changes of PaO2 in living animals, which can provide experimental evidence for explaining the mechanism of switching of inspiration-expiration in HIPM.

[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.