Effect of electroacupuncture pre-conditioning on the expression rhythm of core clock gene Bmal1 in uterine tissue of controlled hyperstimulation rats. / ç”µé’ˆé¢„å¤„ç†å¯¹ä¿ƒæŽ’åµå¤§é¼ å­å®«ç»„ç»‡æ ¸å¿ƒæ—¶é’ŸåŸºå› Bmal1表达节律的影响.

OBJECTIVES: To observe the effect of electroacupuncture (EA) pre-conditioning on the expression rhythm of clock gene Bmal1 in the uterine tissue of rats with controlled ovarian hyperstimulation(COH), so as to explore its mechanisms underlying improvement of the endometrial receptivity of ovarian superovulation during implantation. METHODS: Seventy-two female SD rats with typical estrous cycles were randomly divided into normal control, model and EA pre-conditioning (pre-EA) groups, with 24 rats in each group. The COH model was established by giving the rats with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) by intraperitoneal injection. The rats of the pre-EA group received EA stimulation (1 Hz/5 Hz, a tolerable strength) of "Guanyuan"(CV4) and "Sanyinjiao"(SP6) for 15 min each time, once daily (at 21：00 every day). After successive EA intervention during the first two estrous cycles, the modeling began in the third estrus cycle and the EA intervention was continued till the end of modeling, followed by raising the rats with superovulation induction and male rats undergoing vasoligation in one cage (1∶1). The rats during the estrum in the normal control group or those of the model group at the end of modeling were raised together with the male rats undergoing vasoligation in one cage. On the 5th day (04：00 AM) after raising in one cage, the rats in the three groups were sacrificed in six batches every 4 hours, with 4 rats in each group in each batch. The H.E. staining was used for revealing alterations of the endometrial thickness, number of glands and blood vessels and tissue histology, and ELISA employed to ascertain the contents of estradiol (E2) and progesterone (Pg) in serum. The expression rhythm of core clock gene Bmal1 ［In the present study, Zeitgeber time (ZT) is an artificially set laboratory time, i.e., ZT7 (07：00) is light on and ZT19 (19：00) is light off.］ and the expression of endometrial HoxA10 and leukemia inhibitory factor (LIF) mRNAs were detected by quantitative real-time PCR. The Western blot was employed to detect the expression levels of HoxA10 and LIF proteins. RESULTS: Findings of the clock gene Bmal1 level showed that the expression peak was at ZT12 and the valley value at ZT20 in the normal control group, and that of the peak value was at ZT20 and valley value at ZT12 in the model group, while in the pre-EA group, the peak value was at ZT8, and the valley value at ZT4. The difference of Bmal1 levels among the three groups was most significant at ZT12 (12：00), therefore, the tissue samples were taken at ZT12 in this study for comparison of the levels of different indexes among the 3 groups. Compared with the control group, the endometrial thickness, number of glands and blood vessels, HoxA10 and LIF mRNAs and proteins were significantly down-regulated (P<0.01, P<0.05), and contents of serum E2 and Pg were considerably up-regulated in the model group (P<0.01, P<0.05). Relevant to the model group, the pre-EA group had an apparent increase in the endometrial thickness, number of glands and blood vessels, and expression levels of HoxA10 and LIF mRNAs and proteins (P<0.05, P<0.01), and a marked decrease in the serum Pg (P<0.05). At the ZT12 (12：00 noon), compared with the normal control group, the mRNA level of Bmal1 was significantly decreased in the model group (P<0.01)；and compared with the model group, the level of Bmal1 mRNA was significantly increased in the pre-EA group (P<0.05). In addition, at the node of ZT16, the mRNA level of Bmal1 was significantly decreased in the model group in comparison with the normal control group (P<0.01). CONCLUSIONS: EA preconditioning can improve the endometrial receptivity during the implantation window period in rats with COH, which may be related to its functions in regulating the expression of clock gene Bmal1 in the uterine tissue and in correcting the disturbance of clock gene rhythm.

Synergistic effects of electroacupuncture and bone marrow mesenchymal stem cell transplantation on rat intrauterine adhesion： an observation. / 电针联合骨髓间å è´¨å¹²ç»†èƒžç§»æ¤æ²»ç–—å¤§é¼ å®«è ”ç²˜è¿žçš„ååŒæ•ˆåº”è§‚å¯Ÿ.

OBJECTIVES: To investigate the effects of electroacupuncture(EA) combined with bone marrow mesen-chymal stem cells(BMSCs) transplantation on the endometrium of rats with intrauterine adhesions(IUA), so as to explore the possible mechanisms underlying their combined therapeutic effects. METHODS: Forty adult female SD rats were randomly divided into control, model, cell, and combined groups. The IUA rat model was established using a dual injury method of mechanical scratching and lipopolysaccharide infection. After successful modeling, on days 1, 3, and 7, rats in the model group received tail vein injection of phosphate buffered solution, while rats in the cell group received tail vein injection of BMSCs suspension for BMSCs transplantation；and rats in the combined group received BMSCs transplantation combined with EA treatment (2 Hz/15 Hz, 1-2 mA), targeting the "Guanyuan"(CV4), bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6) for 20 min daily for 3 consecutive estrous cycles. After intervention, uterine tissue was collected from 5 rats in each group. Histological analysis was performed using hematoxylin and eosin staining to evaluate endometrial thickness and glandular number. Masson staining was used to assess endometrial fibrosis area. Immunohistochemistry was performed to detect the positive expressions of vascular endothelial growth factor(VEGF), proliferating cell nuclear antigen(PCNA), and estrogen receptor(ER). Western blot analysis was conducted to determine the protein expressions of homeobox A10(HoxA10) and leukemia inhibitory factor(LIF), both key regulators of endometrial receptivity. The remaining 5 rats in each group were co-housed with male rats, and the uterine function recovery was evaluated by assessing the number of embryo implantations. RESULTS: Compared with the control group, the model group showed thinning endometrium(P<0.001), decreased glandular number(P<0.001), increased endometrial fibrosis area(P<0.001), reduced positive expressions of VEGF, PCNA, ER, expressions of HoxA10 and LIF, and decreased embryo implantation number (P<0.001) on the injured side of the uterus. Compared with the model group, the combined group showed a reversal of the aforementioned indicators(P<0.001, P<0.01)；the cell group exhibited thicker endometrium(P<0.001) and reduced endometrial fibrosis area(P<0.001). Compared with the cell group, the combined group showed increased endometrial thickness(P<0.01), elevated glandular number(P<0.05), significantly decreased endometrial fibrosis area(P<0.05), enhanced positive expressions of VEGF, PCNA and ER, expressions of HoxA10 and LIF in the endometrium, and a significant increase in embryo implantation number (P<0.001, P<0.05, P<0.01) on the injured side of the uterus, indicating better results than the cell group. CONCLUSIONS: The combination of EA and BMSCs synergistically promotes the repair of damaged endometrium, improves endometrial morphology, reduces fibrosis levels, enhances vascular regeneration and matrix cell proliferation, improves endometrial receptivity, which ultimately facilitates embryo implantation.

Antitumor effects of two extracts from Oxytropis falcata on hepatocellular carcinoma in vitro and in vivo.

AIMS: To investigate the antitumor effects of extracts from Oxytropis falcata on human hepatocellular carcinoma SMMC-7721 cells in vitro and in transplanted murine H22 tumors in vivo. METHODS: Cell proliferation, cell cycle distribution and apoptosis in SMMC-7721 cells were determined and tumor growth inhibition in H22 tumors was investigated. Cell cycle distribution was analyzed by flow cytometry with propidium iodide (PI) and Annexin V-FITC/ PI double staining. RESULTS: MTT assay revealed that essential oil and flavonoids of O. falcata (named EOOF and FOF) inhibited proliferation of SMMC-7721 cells in a dose-dependent manner. The IC50 value of EOOF and FOF were 0.115 and 0.097 mg·mL(-1), respectively. Cell cycle was arrested at G(1) phase, and induction of apoptosis occurred in SMMC-7721 cells when subjected to FOF. Growth inhibition in H22 solid tumors transplanted mice was significantly pronounced after being treated with FOF, and the inhibition ratio were 56.1% and 70.8% at the concentration of 30 and 60 mg·kg(-1). CONCLUSION: The results suggest that FOF promotes apoptosis in SMMC-7721 cells and inhibits H22 tumor growth, resulting in a potential antitumor effect on hepatic cancer.

Reversing effects of silybin on TAA-induced hepatic CYP3A dysfunction through PXR regulation.

AIM: Silybin (SB), a major constituent of the milk thistle, has been used to treat several liver disorders. However, liver diseases were always accompanied by CYP450 dysfunction. This study was designed to explore the relationship between the hepatoprotective effect and CYP3A regulation of SB during thioacetamide (TAA)-induced rat liver injury. METHODS: Serum biochemical analysis and histopathological study were taken to evaluate the hepatoprotectinve effect of SB. α-SMA were detected by immunohistochemical analysis and cytokine release in rat liver was determined by ELISA assay. CYP3A and PXR expression were determined by RT-PCR and Western blot analysis, and CYP3A activity was based on the midazolam 4-hydroxylation reaction. Also, siRNA transfection was induced in HepG2 cells to evaluate the effect of PXR on cytotoxicity and CYP3A4 dysregulation caused by TAA. RESULTS: SB showed powerful hepatoprotective effects, and anti-inflammatory and anti-fibrosis effects, and reversed the loss of CYP3A and PXR in TAA-injured rat liver, and decreased PXR translocation into the cell nucleus. PXR silencing weakened the effect of SB on cytoprotection and CYP3A regulation. CONCLUSIONS: PXR was a very important factor of CYP3A regulation and might be the target of SB in TAA-induced liver disease. Also, because of the potential interactions of SB and co-administered medicines, it might be necessary to adjust the dosage in the clinical medication of liver disease.