Optimizing treatment of DNA methyltransferase inhibitor RG108 on porcine fibroblasts for somatic cell nuclear transfer.

Aberration in DNA methylation is believed to be one of the major causes of abnormal gene expression and inefficiency of somatic cell nuclear transfer (SCNT). RG108, a non-nucleoside DNA methyltransferase (DNMT) inhibitor, has been reported to facilitate somatic nuclear reprogramming and improved blastocyst formation. The aim of this study was to investigate interaction effect of RG108 treatment time (24-72 hr) and concentrations (0.05-50 µM) on donor cells, and further to optimize the treatment for porcine SCNT. Our results showed that RG108 treatment resulted in time-dependent decrease of genome-wide DNA methylation on foetal fibroblasts, which only happened after 72-hr treatment in our experiments, and no interaction effect between treatment time and concentration. Remarkable decrease of methylation in imprinted gene H19 and increased apoptosis was observed in 5 and 50 µM RG108-treated cells. Furthermore, the blastocyst rates of SCNT embryos were increased as the fibroblasts treated with RG108 at 5 and 50 µM, and additional treatment during cultivation of SCNT embryos would not provide any advantage for blastocyst formation. In conclusion, the RG108 treatment of 72 hr and 5 µM would be optimized time and concentration for porcine foetal fibroblasts to improve the SCNT embryonic development. In addition, combined treatment of RG108 on donor cells and SCNT embryos would not be beneficial for embryonic development.

Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts.

This study was conducted to evaluate whether the poor developmental capacity of pig embryos after vitrification was related to the occurrence of apoptosis. Parthenogenetic blastocysts were used as the research material. The blastocoel recovery rate, mitochondrial membrane potential (ΔΨm), amount of early apoptosis, activities of several caspases, and relative abundance of mRNA of apoptosis-related genes involved in mitochondria and death receptor apoptotic pathways were detected before or after vitrification. The results indicate that the blastocoel recovery rate (31.0%) and total cells (31.8) of vitrified blastocysts were less than those of fresh blastocysts (100% and 38.2, P < 0.05). The ΔΨm of vitrified blastocysts was 0.46, which was less than that of fresh blastocysts (1.02, P < 0.05). The rate of apoptotic cells in vitrified blastocysts (8.1%) after TUNEL (TdT-mediated dUTP Nick-End Labeling) assay was markedly greater than that in fresh blastocysts (3.9%, P < 0.05). The pan-caspase, caspase-3, caspase-8 and caspase-9 activities of vitrified blastocysts (20.7, 20.6, 17.6 and 19.9) were markedly greater than those of fresh blastocysts (7.4, 6.5, 5.5 and 6.3, P < 0.05). The real-time PCR results indicated that relative abundance of caspase-8 and TNF-α mRNA from death receptor apoptotic pathway and caspase-9 for the mitochondrial apoptotic pathway genes in the vitrified group were greater than those in the fresh group P < 0.05). The relative abundance of Bcl-2 and SOD-1 mRNA for the mitochondrial pathway genes in the vitrified group was less than those in the fresh group (P < 0.05). In conclusion, the poor developmental capacity of vitrified parthenogenetic pig blastocysts was closely related with apoptosis. Both mitochondria and death receptor-mediated apoptotic pathways participated the occurrence of this apoptosis.

Anti-inflammatory effects of tanshinone IIA on atherosclerostic vessels of ovariectomized ApoE mice are mediated by estrogen receptor activation and through the ERK signaling pathway.

AIMS: Estrogen plays a protective role in atherosclerosis. Our preliminary work demonstrated that the active conformation of Tanshinone IIA(TanIIA) is similar to the 17ß-estradiol and it can bind to the estrogen receptor. Here, we hypothesized that Tanshinone IIA might have anti-inflammatory and anti-oxidative effects in atherosclerosis, mediated through estrogen receptor activation. METHODS: Subjects for this study were 120 apoE(-/-) female mice and 20 C57/BL female mice. The apoE(-/-) mice were ovariectomized (OVX) and the C57/BL mice were sham ovariectomized. The sham OVX mice were maintained on a normal diet (NOR) group. The OVX apoE(-/-) mice were fed a high fat diet and randomly divided into 6 groups: Model (MOD) group which was fed a high fat diet only, E2 group were given estrogen (E2) 0.13 mg/kg/d; E2+ICI group were given E2:0.13 mg/kg/d and ICI182780:65 mg/kg/m; TLD group (TanIIA low dose) were given TanIIA: 30 mg/kg/d; THD group (TanIIA high dose) were given TanIIA:60 mg/kg/d; and TLD+ICI group were given TanIIA 30 mg/kg/d and ICI182780 65 mg/kg/m. After three months of treatment, the aorta and the blood of the mice from each group was collected. The aorta were used for testing the lipid deposition by using hematoxylin and eosin(HE) and oil red O staining and for testing the expression of p-ERK1/2 by Western blot. The blood was used for testing the serum cholesterol, superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), nuclear factor kappa (NF-κB), soluble intercellular cell adhesion molecule-1 (sICAM-1), activating protein-1 (AP-1), E-selectin and 17ß-estradiol in serum. RESULTS: Tanshinone IIA significantly reduced the lipid deposition in aorta, decreased the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL), MDA, NF-κB, sICAM-1, AP-1, and E-selectin in serum but increased the levels of high density lipoprotein (HDL) and SOD in serum. Tanshinone IIA also suppressed the expression of p-ERK1/2. Tanshinone IIA had no effect of level of serum 17ß-estradiol levels. All of the effects of Tanshinone IIA were similar to estrogen and were inhibited by the estrogen receptor antagonist ICI182780. CONCLUSION: Tanshinone IIA may play an anti-inflammatory and anti-oxidative stress role in OVX atherosclerotic apoE(-/-) mice by activating the estrogen receptor through the ERK signaling pathway. Therefore, Tanshinone IIA, as a phytoestrogen, could be used for estrogen replacement therapy for cardiovascular disease of postmenopausal women.

Effect of Chinese drugs for activating blood circulation and detoxifying on indices of thrombosis, inflammatory reaction, and tissue damage in a rabbit model of toxin-heat and blood stasis syndrome.

OBJECTIVE: To observe the regulatory effect of Chinese drugs for activating blood circulation (ABC) and for activating blood circulation and detoxifying (ABCD) on indices of thrombosis, inflammatory reaction, and tissue damage in a rabbit model of toxin-heat and blood stasis syndrome. METHODS: Fifty-four rabbits were randomized into the normal control group, model group, simvastatin group (simvastatin, 0.93 mg/kg per day), ABC group [Xiongshao Capsule, 0.07 g/kg per day], and ABCD group [Xiongshao Capsule, 0.07 g/kg per day, and Huanglian Capsule, 0.14 g/kg per day]. All except the normal control group received a single injection of bovine serum albumin and were fed with high-fat diets for 6 weeks. At the end of week 4 of giving high-fat diets, a dose of endoxitin was given by ear vein injection, and a randomized 2-week treatment was initiated. At the end of treatment, blood lipids, circulating endothelial cells, and the pathological changes of the aortic arch were assessed. The serum levels of matrix metalloproteinases (MMP-9), tissue inhibitors to metalloproteinase (TIMP-1), granule membrane protein-140 (GMP-140), plasminogen activator inhibitor-1 (PAI-1), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and tumor necrosis factor-α(TNF-α) were determined. RESULTS: Compared with the model group, ABCD group showed decreased serum triglyceride (TG) level, improvement in the pathological change in the aortic arch, and reduction in the number of circulating endothelial cells (4.00 ± 1.41 per 0.9 µL for ABCD group vs 7.83 ± 1.72 per 0.9 µL for the model group). In addition, the levels of serum GMP-140, PAI-1, and IL-6 in ABCD group were also significantly reduced [0.79 ± 0.20 ng/mL, 5.23 ± 1.39 ng/mL, 40.64 ± 10.11 pg/mL for ABCD group vs 1.08 ± 0.31 ng/mL, 7.28 ± 2.01 ng/mL, 54.44 ± 13.56 pg/mL for the model group, respectively, P < 0.05]. A trend showing improvement in the indices of thrombosis, inflammatory reaction, and tissue damage was observed in the ABC group when compared to the model group, but the changes were not statistically significant (P > 0.05). CONCLUSIONS: Chinese drugs for activating blood circulation and detoxifying have beneficial effects on regulating indices of thrombosis (GMP-140 and PAI-1) and inflammatory reaction (IL-6) in rabbit model with toxic-heat and blood stasis. The effect of the activating blood circulation and detoxifying drugs in regulating the levels of serum GMP-140, PAI-1, and IL-6 was superior to that of the activating blood circulation drugs.

Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus Scrofa).

To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

Correlation between Fc γ R III a and aortic atherosclerotic plaque destabilization in ApoE knockout mice and intervention effects of effective components of chuanxiong rhizome and red peony root.

OBJECTIVE: To explore the correlation between Fc γ RIII A (CD16A) and aortic atherosclerotic plaque destabilization in apoE knockout (apoE KO) mice and the intervention effects of effective components of chuanxiong rhizome and red peony root. METHODS: Eight 8-week-old male C57BL/6J mice were selected as the control group. Forty 8-week-old male apoE KO mice were randomly divided into the model group, apoE KO + intraperitoneal injection immunoglobulin group (IVIG), apoE KO + simvastatin group (Sm), apoE KO + high dosage of xiongshao capsule (XSC) group (XSCH), and apoE KO + low dosage of XSC group (XSCL), 8 mice in each group. Mice in the control group were put on a normal diet, and others were fed with a high-fat diet. After 10-week different interventions, monocyte CD16 expression was detected by flow cytometry, aortic matrix metalloproteinase-9 (MMP-9) mRNA expression was detected using reverse transcription polymerase chain reaction, and serum tumor necrosis factor (TNF)-α level was detected using enzyme-linked immunosorbent assay. RESULTS: Compared with the control group, monocyte CD16 expression, aortic MMP-9 mRNA expression, and serum TNF-α level in the model group increased obviously (P<0.01). Injections of apoE KO mice with intraperitoneal immunoglobulin during a 5-day period significantly reduced the monocyte CD16 expression, aortic MMP-9 mRNA expression, and serum TNF-α level (P<0.01 or 0.05) over a 10-week period of high-fat diet. Indices above in the Sm group, XSCH group, and XSCL group decreased in a different degree. Of them, the aortic MMP-9 mRNA expression in XSCH group was lower than that in Sm group (P<0.05) and the monocyte CD16 expression and serum TNF-α level showed no significant difference between XSCH group and Sm group (P>0.05). Correlation analyses suggested positive correlation between monocyte CD16 expression and aortic MMP-9 mRNA expression or serum TNF-α level in IVIG group, XSCH group, and XSCL group. CONCLUSIONS: FcγR III A mediates systemic inflammation in the progression of coronary heart disease with blood stasis syndrome. XSC could stabilize atherosclerotic plaque by suppressing inflammation and its target was relative with FcγRIII A.

[Effects of Chinese herbal drug-containing serum on oxidative damage and apoptosis of umbilical vein endothelial cells induced by oxidized low-density lipoprotein].

OBJECTIVE: To investigate the effects of Chinese herbal drug-containing serum, prepared by administration of Chinese herbal medicine for activating blood (Xiongshao Capsule, XS) or for activating blood and detoxifying (Xiongshao Capsule plus Huanglian Capsule, XSHL) in rats, on cell viability, oxidative damage and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL). METHODS: Thirty-two rats were randomly divided into 4 groups: control group, positive control group (simvastatin 1.8 mg/kg), activating blood (XS, 0.135 g/kg) group, and activating blood and detoxifying (XS Capsule 0.135 g/kg and Huanglian Capsule 0.135 g/kg, XSHL) group. Corresponding drugs were continuously administered to the rats for 7 days and then drug-containing serum was harvested 1 hour after the last administration. HUVECs isolated from newborn children by collagenase digestion were stimulated by ox-LDL (100 µg/mL) [corrected] and incubated with corresponding drug-containing serum for 24 hours. Untreated HUVECs were also used as a normal control. The morphology and structure of HUVECs were observed by an inverted microscope. Cell viability was measured by methyl thiazolyl tetrazolium method, and cell membrane damage was determined by lactate dehydrogenase (LDH) leakage. Activity of superoxide dismutase (SOD) was examined by spectrophotometry, and content of malondialdehyde (MDA) in the cell lysate was examined by thiobarbituric acid assay. HUVECs were stained with Annexin V-fluorescein isothiocyanate and propidium iodide and analyzed on a flow cytometry to determine apoptosis. RESULTS: Compared with the normal HUVECs, the cell viability and the activity of SOD were significantly decreased while the content of MDA and apoptosis rate were significantly increased after 24-hour ox-LDL stimulation (P<0.01, P<0.05). Simvastatin-, XS-, and XSHL-containing serum significantly promoted the ox-LDL-stimulated HUVEC viability and inhibited early apoptosis (P<0.01, P<0.05), while had no significant effect on LDH leakage. Simvastatin-containing serum and XS-containing serum also showed significant decrease in MDA content and increase in SOD activity, while XSHL-containing serum showed no significant effects. There was no significant difference between the XS-containing serum group and the XSHL-containing serum group. CONCLUSION: Both sera containing XSHL and XS show protective action against the oxidative damage and apoptosis of HUVECs induced by ox-LDL.

Synergistic protection of Danhong injection (丹红注射液) and ischemic postconditioning on myocardial reperfusion injury in minipigs.

OBJECTIVE: To explore the synergistic protection of Danhong Injection (丹红注射液, DHI) and ischemic postconditioning on myocardial reperfusion injury in minipigs. METHODS: Acute myocardial infarction model was made by balloon occlusion in left anterior descending coronary artery (LAD) of minipigs, and then postconditioning was simulated through inflation/deflation of the angioplasty balloon. Minipigs were divided into four groups: the sham operation group (SH group), the ischemia/reperfusion group (I/R group), the ischemic postconditioning group (POC group) and DHI combined with ischemic postconditioning group (PAD group, DHI 20 mL through ear vein), six in each group. After 24-h continuous observation, myocardial infarction size was assessed by triphenyltetrazolium staining (TTC). Morphological changes of ischemic myocardium were observed by light microscopy, and cardiomyocyte ultrastructure was studied with electron microscopy. The superoxide dismutase (SOD) and malondialdehyde (MDA) activity in heart homogenates were measured by a biochemical method. RESULTS: The myocardial infarction size was smaller in the POC group than in the I/R group (0.26 ± 0.02 vs. 0.37 ± 0.09, P<0.05), and the PAD group (0.14 ± 0.08) displayed a significantly reduced infarction size relative to the I/R group (P<0.01) and POC group (P<0.05). The damage of myocardial tissue was severe in the I/R group shown by light and electron microscopy: myocardial fibers disorder, sarcoplasmic dissolution, myofilament fracture, mitochondria swelling and even vacuolization formation and a large number of inflammatory cell infiltrations. Compared with the I/R group, reduction of reperfusion injury in the PAD group included more orderly arranged myocardial fibers, less infiltration of inflammatory cells and maintenance of mitochondrial integrity. Compared with the I/R group, the damage of myocardial tissue in the POC group was improved, but not as significant as that in the PAD group. SOD levels in the POC group and the PAD group were significantly higher than those in the I/R group (96.96 ± 13.43, 112.25 ± 22.75 vs. 76.32 ± 10.63, P<0.05), and MDA was significantly lower in the POC group and the PAD group compared to the I/R group (1.27 ± 0.19, 1.09 ± 0.21 vs. 1.47 ± 0.16, P<0.05). CONCLUSION: DHI and ischemic postconditioning show a synergistic cardioprotection on myocardial reperfusion injury in minipigs.

Effects of active components of Red Paeonia and Rhizoma chuanxiong on angiogenesis in atherosclerosis plaque in rabbits.

OBJECTIVE: To investigate the effects and mechanism of the active components of Red Paeonia and Rhizoma chuanxiong (Xiongshao Capsule, XSC) on angiogenesis in atherosclerosis plaque in rabbits. METHODS: Fifty New Zealand rabbits were randomly divided into the normal group, the model group, and the three medicated groups treated respectively with Simvastatin (2.5 mg/kg per day), low-dose (0.24 g/kg per day) and high-dose (0.48 g/kg per day) XSC, 10 in each group. Rabbits in the normal group were fed with regular diet. To those in the other four groups, high fat diet was given, and a balloon angioplasty was performed two weeks later to establish abdominal aortic atherosclerosis model. Then, the model rabbits were fed continuously with high fat diet, and to those in the medicated groups, the testing drugs were added in the forage correspondingly for 6 successive weeks. Levels of blood lipids were measured at the end of the experiment. Meantime, serum levels of high sensitivity C-reactive protein (hsCRP) and tumor necrosis factor alpha (TNF-alpha) were detected with enzyme-linked immunoassay; the plaque area (PA), cross-sectional vascular area (CVA) and correcting plaque area (PA/CVA) were determined quantitatively using imaging software; and the protein expression of vascular endothelial growth factor (VEGF) and factor VIII related antigen (FVIIIRAg) in plaque was detected using immunohistochemical method. RESULTS: As compared with the model group, the content of total cholesterol (TC) in the three medicated groups, and contents of triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the Simvastatin group were lower to various extents (P<0.05, P<0.01). The serum level of hsCRP in all modeled rabbits was higher than that in the normal group, but in the three treated groups it was significantly lower than that in the model group (P<0.05, P<0.01). Expressions of VEGF and FVIIIRAg, as well as PA/CVA in the three medicated groups were significantly lower than those in the model control group (P<0.05, P<0.01). CONCLUSION: The active components of Red Paeonia and Rhizoma chuanxiong have definite effects in delaying the genesis and development of atherosclerosis, its mechanism might be related with the inhibition on angiogenesis in plaque, and also with its actions of lipo-metabolism regulation and anti-inflammation.

[Effects of 5 ml straws cryopreservation on the quality and ultrastructure of boar semen].

OBJECTIVE: To investigate damages to the quality of boar sperm frozen in 5 ml straws, pellet and 0.25 ml straws as well as the ultrastructural changes of frozen boar sperm in 5 ml straws. METHODS: We compared 3 different freezing packages of 5 ml straws, pellet and 0.25 ml straws to determine their effects on frozen boar semen, and observed the morphological and ultrastructural changes of the boar sperm in the 5 ml straws using scanning electron microscopy. RESULTS: In the 5 ml straws, the vitality and motility of the boar sperm after freezing were not significantly different from those in the other two formulations, the normal apical ridge (NAR) was 52.65%, higher than in the pellet but not significantly different from that in the 0.25 ml straws, and the sperm membranes were mostly bubbly, some locally broken, which indicated the damage induced by freezing and thawing. CONCLUSION: At the present time, boar semen frozen in 5 ml straws were not significantly different from those frozen in 0.25 ml straws. The existing freezing-thawing method may cause certain damage to the quality and ultrastructure of boar sperm, and therefore needs to be further improved.

[Chromosome changes of aged oocytes after ovulation].

In mouse, matured oocytes are arrested at metaphase- (M) after ovulation. If the M oocytes are not fertilized on time, they will increasingly become aged in the oviduct. The aging of oocytes can lead to abnormal development of embryos. So it is important to study the mechanism of oocyte aging. Herein, we studied the change of oocyte chromosome after ovulation into the oviduct in vivo. Results indicated that 65% of old oocytes (34 h post hCG) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to the young oocytes (14 h post hCG). In addition, chromosome changes may be associated with the increase of acetylation of histone 3 and histone 4, at lysine 14 and lysine 16 (H3K14 and H4K16), respectively. On the other hand, the decrease of methylation of histone 3 at lysine 9 (H3K9) presumably facilitated aberrant chromosome formation.