[Mechanism of Liangfang Wenjing Decoction in treatment of hypoxia on endometriosis with cold coagulation and blood stasis by regulating CHCHD4 expression].

To explore the mechanism of Liangfang Wenjing Decoction regulating coiled-coil-helix coiled-coil-helix domain containing 4(CHCHD4) in the treatment of hypoxia on endometriosis(EMs) with cold coagulation and blood stasis. The rat model of cold coagulation and blood stasis syndrome was prepared by the ice-water bath method, and then the EMs model was established by autologous intimal transplantation. The rats were randomly divided into model group, low, medium, and high(4.7, 9.4, and 18.8 g·kg~(-1)) dose groups of Liangfang Wenjing Decoction, Shaofu Zhuyu Decoction group, and sham group, with 10 rats in each group. The rats were given intragastric administration for four weeks. During the modeling, the general condition and vaginal smear of rats were observed, and the blood flow of ears and uterus were detected by laser speckle contrast imaging(LSCI) to judge the syndrome of cold coagulation and blood stasis. After the administration, the general condition of the rats was observed, and the area of ectopic lesions was measured by caliper. The localization and expression of CHCHD4 and hypoxia inducible factors-1α(HIF-1α) were detected by immunohistochemistry, and the mRNA and protein expressions of CHCHD4 and HIF-1α were detected by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot. The primary culture of ectopic endometrial stromal cells(ESCs) from EMs patients was performed, and the CHCHD4 overexpression plasmid was constructed and transfected to establish the ESCs model of CHCHD4 overexpression. The cells were divided into the control group, CHCHD4 overexpression group, CHCHD4 overexpression+control serum group, and CHCHD4 overexpression+Liangfang Wenjing Decoction serum group. The protein expression of CHCHD4 and HIF-1α was detected by Western blot, and the glucose consumption and lactic acid level were detected. The cell proliferation was detected by MTT assay. The experiment found that compared with normal rats, the modeling rats showed symptoms of cold coagulation and blood stasis, such as mental malaise, reduced diet and drinking water, disordered estrous cycle, and blocked blood circulation in ears and uterine microvessels. Compared with the sham group, the ectopic lesions in the model group were uplifted, and the mRNA and protein expressions of CHCHD4 and HIF-1α were significantly increased(P<0.05). Compared with the model group, the symptoms of cold coagulation and blood stasis in each treatment group were improved, and the area of ectopic lesions was significantly reduced(P<0.05 or P<0.01). The mRNA and protein expression levels of CHCHD4 and HIF-1α were significantly decreased(P<0.05 or P<0.01). In the cell model, compared with the control group, the expression of CHCHD4, HIF-1α protein, glucose consumption, lactic acid level, and cell proliferation activity in the CHCHD4 overexpression group were significantly increased(P<0.01). Compared with the CHCHD4 overexpression group, there was no significant change in each index in the control serum group, while the protein expression of CHCHD4 and HIF-1α in the Liangfang Wenjing Decoction serum group was decreased significantly(P<0.05 or P<0.01). The glucose consumption, lactic acid level, and cell proliferation activity decreased significantly(P<0.01). It can be seen from the above that the therapeutic effect of Liangfang Wenjing Decoction on EMs with cold coagulation and blood stasis might be related to reducing the expression of CHCHD4 and then improving the hypoxia of ectopic lesions and ectopic ESCs.

[Effect of Liangfang Wenjing Decoction on expression of key glycolytic enzymes in uterus and ovaries of rats with coagulating cold and blood stasis syndrome].

This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.

[Establishment of gynecological asthenia cold syndrome model].

OBJECTIVE: To establish the model of gynecological asthenia cold syndrome by simulating the etiology of gynecological asthenia cold syndrome depending on 'pathogenic cold impairing yang" in Chinese medicine theory. METHODS: The female SD rats were randomly divided into the model group and the normal group by randomized digital table, 20 in each group. Rats in the model group were placed in 0 degrees C - 1 degree C ice water, and the ice water was placed in 4 degrees C refrigerator, twice daily, 20 min each time, for a total of 30 days. The body temperature was determined and the changes of the estrous cycle were observed every day. When the body temperature decreased (with statistical difference from those of the normal group), and vaginal smears showed disordered estrous cycle, the model was successfully established. Rats were sacrificed during the diestrus period, the correlative indices including reproductive endocrine hormones in blood [follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol ( E2 ), testosterone (T), and progestone (P)], the thyroid function [triiodothyronine (T3), tetraiodothyronine (T4), thyrotropic-stimulating hormone (TSH)], the adrenal function [plasma adrenocorticotropic hormone (ACTH) and cortisol (Cor)], the cellular immune function [serum interleukin-2 (IL-2)], and energy tests [including plasma cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), the ratio of cAMP/cGMP, lactate dehydrogenase(LDH)], an the index of thymus, spleen, uterus, and ovary were detected. RESULTS: Compared with the normal group, rats in the model group appeared chill, dim-color hair, purple and dark ears and claw. They were depressed, scrunched, quiet, and clumsy. They liked to stay together. Their water intake and appetite were reduced, body weight lost, body temperature significantly lowered. They passed loose stool. Their estrous cycle and diestrus were prolonged. Their plasma cGMP content, cAMP/cGMP ratio, LDH, serum IL-2 content, E2, P, T, T3, LH, TSH were significantly lowered to some extent. Their thymus index and the ovary index significantly decreased, showing significant difference (P < 0.05). CONCLUSION: The rat model of gynecological asthenia cold syndrome prepared by extending the frozen time at refrigerator and ice water immersion was in line with clinical features of gynecological asthenia cold syndrome.

Effects of cullin 4B on the proliferation and invasion of human gastric cancer cells.

The major aim of the present study was to explore the effects of cullin 4B (CUL4B) on the proliferation and invasion of human gastric cancer cells. Gastric tumor tissues and paired adjacent non­tumor tissues were obtained from 21 gastric cancer patients, and gastric cancer cell lines (AGS, MGC­803, KATO­III, MKN­45, SGC­7901, BGC­823 and MKN­74) were cultured. BGC­823 cells were transfected with CUL4B small interfering (si)RNA or control siRNA. Reverse transcription­quantitative polymerase chain reaction analysis was performed to detect the mRNA expression of CUL4B. Western blot analysis was performed to measure the protein levels of Wnt, ß­catenin, glutathione synthase kinase (GSK)­3ß, caspase­3 and cyclin E. MTT and Transwell assays were performed to examine cell proliferation and invasion following CUL4B knockdown. In addition, the effect of CUL4B knockdown on the cell cycle and apoptosis of BGC­823 cells was evaluated by flow cytometric analysis. The results indicated that compared with the adjacent non­tumor tissues and a normal gastric epithelial cell line, gastric cancer tissues and cell lines exhibited significantly higher expression of CUL4B. Knockdown of CUL4B in gastric cancer cells suppressed cell proliferation, caused G1 arrest and inhibited cell invasion. Silencing of CUL4B also resulted in decreased Wnt and ß­catenin expression, but increased expression of GSK­3ß, caspase­3 and cyclin E. These results indirectly demonstrate that CUL4B enhances the proliferation and invasion abilities of gastric cancer cells by upregulating the constituent factors Wnt and ß­catenin, as well as by negatively regulating the mRNA and protein expression of GSK­3ß, caspase­3 and cyclin E. The potential mechanism of CUL4B highlighted in the present study may be helpful for the treatment of patients with gastric cancer.

Binary competitive adsorption of naphthalene compounds onto an aminated hypercrosslinked macroporous polymeric adsorbent.

An aminated hypercrosslinked macroporous polymeric adsorbent was synthesized and characterized. Adsorption isotherms for 1-amino-2-naphthol-4-sulfonic acid (1,2,4-acid) and 2-naphthol obtained from various binary adsorption environments can be well fitted by Freundlich equation, which indicated a favorable adsorption process in the studied range. Adsorption for 1,2,4-acid was an endothermic process in comparison with that for 2-naphthol of an exothermic process. 2-naphthol molecules put a little influence on the adsorption capacity for 1,2,4-acid. However, the adsorption to 1,2,4-acid depressed that to 2-naphthol in a large extent for the stronger electrostatic interaction between 1,2,4-acid and adsorbent. The predominant mechanism can be contributed to the competition for adsorption sites. And the simultaneous environment was confirmed to be helpful to the selective adsorption towards 1,2,4-acid based on the larger selectivity index.