Background: From the perspective of Mongolian medicine, hemorrhagic disease is a symptom of bleeding from any part of the body. This disease was compared to the immune thrombocytopenia disease of modern medicine. The treatment of this disease using two medical methods and the prevention of complications and relapses are issues facing the healthcare sector. In this regard, we have chosen this topic to clarify and prove the mechanism of action of the Mongolian drug Gurgem-8, which is widely used to treat bleeding disorders. Aim: To evaluate the therapeutic efficacy of Gurgem-8, in haemostatic treatment. Materials and Methods: The study was conducted using a randomized, controlled (active), open label, single centered clinical trial method. The study was conducted in two phases. First, an acute toxicity study of the Gurgem-8, was conducted in accordance with OECD guideline 423 and evaluated according to GHS classification. A chronic toxicity study was also conducted on Wistar rats (n=20) given the Gurgem-8, at doses of 500 mg/kg, 100 mg/kg, and 150 mg/kg daily for 60 days. Second, a clinical study was conducted on a total of 74 patients, who were randomly divided into 2 groups. The treatment group was given 3 grams of the Gurgem-8, daily, and the comparison group was given 4 capsules of Sheng Xue Xiao Ban Jiao Nang 3 times a day. The results were determined by laboratory methods. The study was conducted with the approval of the Research Ethics Committee of Mongolian National University od Medical Sciences (2024.01.19 №2024/3-01). Results: In the acute toxicity study, Turmeric-8 was found to be of low toxicity according to the GHS classification. No mortality was observed in the chronic toxicity test. As a result of the clinical study, there were significant differences in the blood hemoglobin (χ2=73.923, P<0.001), platelet (χ2=62.465, P<0.001), erythrocyte (χ2=77.113, P<0.001) and leukocyte (χ2=14.771, P<0.001) cell counts between the Gurgem-8, drug group and the comparison group. It was also determined that the platelet (χ2=138.3, P<0.001), erythrocyte (χ2=85.405, P<0.001) and leukocyte (χ2=10.961, P=0.027) cell counts were directly related to the treatment period and the group. When determining the effect on immune cells, there was no significant difference in the lymphocyte cell content before and after treatment (CD4+: t=0.233, P=0.817; CD8+: t=-0.264, P=0.793; CD4/CD8:Z=-0.119, P=0.905). However, the CD4/CD8 ratio was statistically significantly increased in each of the Gurgem-8, drug group and the comparison group (P<0.001, P=0.001). Conclusion In immune thrombocytopenia diseases, the Gurgem-8, has the effect of reducing hemoglobin levels in the blood, increasing platelet counts, reducing CD4+ and CD8+ T cell counts, and increasing the CD4/CD8 ratio.

Objective: To investigate the correlation between CpG islands methylation statuses of TGF-β₃, Dnmts and their expression during TCDD-induced mouse embryonic palatal development. Methods: Eithtteen pregnant C57BL/6J mice were randomly divided into 2 groups: the control group(n=9) and TCDD-exposure group(n=9). At gestation day 10.5(GD10.5), the mice in TCDD-group were orally administrated with TCDD 28 μg/kg, while the mice in the control group received equivalent corn oil. The pregnant mice were sacrificed at GD13.5, GD14.5, GD15.5, fetal palates were collected. CpG island methylation statuses were analysed by methylation specific polymerase chain reaction(MSP). IBM SPSS 20.0 software was applied for statistical analysis. Kolmogorov-Smirnov test was used for normal distribution check, and the distributions were normal. Independent t-test was carried out between two groups. P<0.05 was considered statistically significant. Results: CpG island in promoter region of gene TGF-β₃ were all at low methylation level at all GDs of both groups, there were no differences at same GD between two groups [GD13.5: (8.6±0.8)% vs (8.7±0.8)%, P>0.05; GD14.5: (11.5±1.4)%vs (11.7±1.0)%, P>0.05; GD15.5: (12.0±0.7)% vs (12.1±0.5)%, P>0.05]. CpG island in promoter region of gene Dnmt1 were all highly methylated with no differences showed at same GD between two groups [GD13.5: (73.9±1.1)%vs (72.6±0.8)%, P>0.05; GD14.5: (70.8±1.7)% vs (70.7±1.0)%, P>0.05; GD15.5: (69.4±2.2)% vs (69.7±0.5)%, P>0.05]. The methylation level of CpG island 1 in promoter region of gene Dnmt3a in TCDD group was higher than that in control group at GD13.5 and GD15.5 [(21.9±1.1)% vs (8.1±0.6)%, P<0.01, (43.4±0.4)% vs(32.9±0.7)%, P<0.01], while lower at GD14.5[(33.2±0.5)% vs (42.9±0.3)%, P<0.01]. The methylation level of CpG island 2 in promoter region of gene Dnmt3a in control group was higher than that in TCDD group at all GDs [GD13.5: (82.0±0.7)% vs (32.3±0.6)%, P<0.01; GD14.5: (62.7±1.0)%vs (25.5±1.4)%, P<0.01; GD15.5: (47.2±0.4)% vs (30.3±1.4)%, P<0.01]. Conclusions Low methylation level of CpG island 2 which is close to the first exon in promoter region of gene Dnm3a may be the cause of highly expressed Dnmt3a mRNA at GD13.5 during mice palatogenesis induced by TCDD, thus the global DNA methylation is extremely high to induce cleft palate. TCDD-treatment doesn′t influence the CpG methylation statuses in promoter region of TGF-β₃ and Dnmt1.

Objective: To explore the common differentially expressed proteins in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin(TCDD) and retinoic acid-induced cleft palate of fetal mice by isobaric tags for relative and absolute quantitation(iTRAQ) combined with mass spectrometry. Methods: Thirty-six pregnant C57BL/6J mice were randomly divided into 3 groups, 12 cases in each group. C57BL/6J pregnant mice were given a gavage of TCDD 28 μg/kg or retinoic acid 80 mg/kg on gestational day 10.5(GD10.5) as experimental groups, while the control group received equivalent corn oil. Anatomical and histological changes of palates in fetal mice were observed on GD17.5. Total proteins were extracted from palates of fetal mice in each group on GD17.5. Differentially expressed proteins were identified in experimental groups as well as in control group by iTRAQ combined with two-dimensional liquid chromatography/tandem mass spectrometry. Western Blot was used for validation of the differentially expressed proteins of Annexin A1 and 14-3-3 sigma. All statistical analyses were measured with SPSS software(version 17.0). Chi-square test was used to compare the incidence of cleft palate. One-way ANOVA was carried out for comparison of the relative expression levels of three groups, homogeneity of variance was analyzed by Levene test, and Turkey HSD test was used for comparison between two groups. P values were judged as significant difference if they were less than 0.05. Results: ①Model of cleft palate in fetal mice were successfully established with incidence of cleft palate of 97.1%(68/70)in TCDD group and 98.6%(70/71) in retinoic acid group, respectively(χ²=0.00, P>0.05), without significant difference between two groups. However, they were similar on the phenotype. ② A total of 2 996 proteins were identified. Compared with control group, 75 and 90 differentially expressed proteins were screened out from TCDD group and retinoic acid group respectively. There were 18 differentially expressed proteins in common both in two experimental groups. ③Western Blot assay indicated that the expression of Annexin A1 protein was 0.52±0.11 in control group, while in TCDD group was 0.99±0.34 and in retinoic acid group was 0.98±0.31, with significant difference between any of two experimental groups and control group(P<0.05). The expression of 14-3-3 sigma protein in control group was 0.55±0.15, while in TCDD group was 0.86±0.17 and in retinoic acid group was 0.93±0.13, with significant difference between any of two experimental groups and control group(P<0.05). These results were consistent with the results of iTRAQ experiment. Conclusions Using iTRAQ technology can quickly and effectively filtrate the common differentially expressed proteins in fetal mice with cleft palate induced by TCDD and retinoic acid. These proteins may have closely related relationship with the occurrence of cleft palate induced by TCDD or retinoic acid.

Objective To investigate global DNA methylation and DNA methyhransferases participation in the mechanism of cleft palate induced by maternal exposure to 2,3,7,8-tetrachlrodibenzo-p-dioxin (TCDD)in mice.Methods 40 pregnant C57BL/6J mice were randomly divided into 2 groups:the control group(n =20) and TCDD-exposure group(n =20).On gestation day 10.5 (GD10.5),the mice in TCDD-group were orally administrated with TCDD 28 μg/kg,while the mice in the control group received equivalent corn oil.The pregnant mice were sacrificed on GD13.5,GD14.5,GD15.5,GD16.5,GD17.5,fetal palates were collected for analysis.Global DNA methylation levels were detected by MethylampTM Global DNA Methylation Quantification Ultra Kit through an ELISA-like reaction.The expression levels of DNA methyltransferases were examined by quantitative real-time PC R(q-PCR).IBM SPSS 20.0 software was applied for statistical analysis.Kolmogorov-Smirnov test was used for normal distribution check,and the distribution was normal.Independent t-test was carried out among two groups.P ＜ 0.05 was considered statistically significant.Results The global DNA methylation level in TCDD-exposure group was significantly higher than that in control group on GD13.5 (49.52％ ±4.03％ vs 33.42％ ± 6.78％,P ＜ 0.01),whilelower on GD14.5 (24.10％ ±2.29％ vs 30.12％ ±3.92％,P ＜0.05) and on GD16.5 (32.77％ ±0.98％ vs 36.45％ ± 3.27％,P ＜ 0.05).The expression level of Dnmt1 mRNA in TCDD-exposure group was higher than that in control group on GD13.5(1.28±0.11 vs 1.01 ±0.10,P＜0.05) and on GD16.5(1.04 ±0.05 vs 0.81 ±0.01,P ＜0.01).The expression level of Dnmt3a mRNA in TCDD-exposure group was higher than that in control group on GD13.5 (1.15 ±0.17 vs 0.81 ±0.02,P ＜0.05)and on GD16.5 (1.11 ± 0.06 vs 0.96 ± 0.06,P ＜ 0.05).The expression level of Dnmt3b mRNA in TCDD-exposure group was higher than that in control group on GD14.5(0.97 ±0.06 vs 0.72 ±0.06,P ＜0.01).Conclusions It is supposed that complicated mechanisms are exist to regulate global DNA methylation levels in palatal tissue of fetal mice.The significant increased DNA methylation level on GD13.5 resulting from up-expression of Dnmt1 and Dnmt3a may be one of the epigenetic mechanisms which cause palate malformation in fetal mice induced by maternal exposure to TCDD.

Objective To investigate global DNA methylation and DNA methyhransferases participation in the mechanism of cleft palate induced by maternal exposure to 2,3,7,8-tetrachlrodibenzo-p-dioxin (TCDD)in mice.Methods 40 pregnant C57BL/6J mice were randomly divided into 2 groups:the control group(n =20) and TCDD-exposure group(n =20).On gestation day 10.5 (GD10.5),the mice in TCDD-group were orally administrated with TCDD 28 μg/kg,while the mice in the control group received equivalent corn oil.The pregnant mice were sacrificed on GD13.5,GD14.5,GD15.5,GD16.5,GD17.5,fetal palates were collected for analysis.Global DNA methylation levels were detected by MethylampTM Global DNA Methylation Quantification Ultra Kit through an ELISA-like reaction.The expression levels of DNA methyltransferases were examined by quantitative real-time PC R(q-PCR).IBM SPSS 20.0 software was applied for statistical analysis.Kolmogorov-Smirnov test was used for normal distribution check,and the distribution was normal.Independent t-test was carried out among two groups.P ＜ 0.05 was considered statistically significant.Results The global DNA methylation level in TCDD-exposure group was significantly higher than that in control group on GD13.5 (49.52％ ±4.03％ vs 33.42％ ± 6.78％,P ＜ 0.01),whilelower on GD14.5 (24.10％ ±2.29％ vs 30.12％ ±3.92％,P ＜0.05) and on GD16.5 (32.77％ ±0.98％ vs 36.45％ ± 3.27％,P ＜ 0.05).The expression level of Dnmt1 mRNA in TCDD-exposure group was higher than that in control group on GD13.5(1.28±0.11 vs 1.01 ±0.10,P＜0.05) and on GD16.5(1.04 ±0.05 vs 0.81 ±0.01,P ＜0.01).The expression level of Dnmt3a mRNA in TCDD-exposure group was higher than that in control group on GD13.5 (1.15 ±0.17 vs 0.81 ±0.02,P ＜0.05)and on GD16.5 (1.11 ± 0.06 vs 0.96 ± 0.06,P ＜ 0.05).The expression level of Dnmt3b mRNA in TCDD-exposure group was higher than that in control group on GD14.5(0.97 ±0.06 vs 0.72 ±0.06,P ＜0.01).Conclusions It is supposed that complicated mechanisms are exist to regulate global DNA methylation levels in palatal tissue of fetal mice.The significant increased DNA methylation level on GD13.5 resulting from up-expression of Dnmt1 and Dnmt3a may be one of the epigenetic mechanisms which cause palate malformation in fetal mice induced by maternal exposure to TCDD.

[摘 要] 目的：探讨1, 25-二羟维生素D3（VD3）对人乳腺癌MCF-7细胞凋亡的影响及其作用机制。方法：取体外培养的人乳腺癌MCF-7细胞，随机分为6组：对照组、2-脱氧葡萄糖（2-DG，葡萄糖抑制剂）组、1 µmol/L VD3组、10 µmol/L VD3组、2-DG+1 µmol/L VD3组和2-DG+10 µmol/L VD3组。药物干预6组细胞48 h后，以葡萄糖摄取测定试剂盒检测细胞的葡萄糖摄取量、ATP试剂盒检测细胞中ATP含量和乳酸试剂盒检测细胞的乳酸水平，WB法检测MCF-7细胞中细胞色素C（Cyt c）和凋亡相关蛋白（Bcl-2、BAX、PARP1、caspase9和caspase3）的表达水平。结果：与对照组比较，VD3干预后，MCF-7细胞的凋亡率明显增加（P<0.05或P<0.01），同时细胞的葡萄糖摄取量、ATP含量及乳酸水平均明显降低（P<0.05或P<0.01），Cyt c、BAX、PARP1、caspase9及caspase3蛋白表达量明显升高（均P<0.05），Bcl-2蛋白表达量降低（P<0.05或P<0.01）；VD3联合2-DG干预后，各组细胞检测指标的变化更为明显（P<0.05或P<0.01）。结论：VD3可通过抑制人乳腺癌MCF-7细胞的糖酵解过程并以线粒体的Cyt c途径促进细胞凋亡。